Increased isoprene production using the archaeal lower mevalonate pathway

ABSTRACT

The invention features methods for producing isoprene from cultured cells using a feedback-resistant mevalonate kinase polypeptide, such as an archaeal mevalonate kinase polypeptide. The resulting isoprene compositions may have increased yields and/or purity of isoprene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claims the benefit of U.S. Provisional patentapplication 61/097,186, filed on Sep. 15, 2008, and U.S. Provisionalpatent application 61/187,876, filed on Jun. 17, 2009, the contents ofboth are hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Isoprene (2-methyl-1,3-butadiene) is the critical starting material fora variety of synthetic polymers, most notably synthetic rubbers.Isoprene is naturally produced by a variety of microbial, plant, andanimal species. In particular, two pathways have been identified for thebiosynthesis of isoprene: the mevalonate (MVA) pathway and thenon-mevalonate (DXP) pathway (FIGS. 19A and 19B). However, the yield ofisoprene from naturally-occurring organisms is commerciallyunattractive. About 800,000 tons per year of cis-polyisoprene areproduced from the polymerization of isoprene; most of this polyisopreneis used in the tire and rubber industry. Isoprene is also copolymerizedfor use as a synthetic elastomer in other products such as footwear,mechanical products, medical products, sporting goods, and latex.

Currently, the tire and rubber industry is based on the use of naturaland synthetic rubber. Natural rubber is obtained from the milky juice ofrubber trees or plants found in the rainforests of Africa. Syntheticrubber is based primarily on butadiene polymers. For these polymers,butadiene is obtained as a co-product from ethylene and propylenemanufacture.

While isoprene can be obtained by fractionating petroleum, thepurification of this material is expensive and time-consuming. Petroleumcracking of the C5 stream of hydrocarbons produces only about 15%isoprene. Thus, more economical methods for producing isoprene areneeded. In particular, methods that produce isoprene at rates, titers,and purity that are sufficient to meet the demands of a robustcommercial process are desirable. Also desired are systems for producingisoprene from inexpensive starting materials.

The invention provided herein fulfills these needs and providesadditional benefits as well.

BRIEF SUMMARY OF THE INVENTION

The invention provides for, inter alia, compositions and methods forincreasing the production of isoprene by using feedback-resistantmevalonate kinase, such as those found in archaeal organisms. In someembodiments, the invention provides for cells comprising (i) one or morenon-modified nucleic acids encoding feedback-resistant mevalonate kinasepolypeptides or (ii) one or more additional copies of an endogenousnucleic acid encoding a feedback-resistant mevalonate kinasepolypeptide. In some embodiments, the feedback-resistant archaealmevalonate kinase polypeptide is M. mazei mevalonate kinase. In someembodiments, the cells in culture comprise (i) a heterologous nucleicacid encoding a feedback-resistant mevalonate kinase polypeptide (e.g.,a Methanosarcina mazei mevalonate kinase polypeptide or a homologthereof) and/or (ii) a duplicate copy of an endogenous nucleic acidencoding a feedback-resistant mevalonate kinase polypeptide (e.g., aMethanosarcina mazei mevalonate kinase polypeptide or a homologthereof). In some embodiments, the mevalonate kinase nucleic acid isoperably linked to a promoter. In some embodiments, thefeedback-resistant mevalonate kinase polypeptide is an archaealmevalonate kinase polypeptide (e.g., a Methanosarcina mazei mevalonatekinase polypeptide). In some embodiments, concentrations of geranyldiphosphate (GPP) or farnesyl diphosphate (FPP) that are equal to orless than about any of 20, 30, 40, 50, 60, 70, 80, 90, or 100 μM do notsubstantially inhibit (e.g., inhibit by less than about any of 10, 8, 6,4, 3, 2, 1%) the ability of the feedback-resistant mevalonate kinasepolypeptide to bind ATP, bind mevalonate, or convert mevalonate tomevalonate-5-phosphate. In some embodiments, concentrations of3,3-dimethylallyl diphosphate (DMAPP) that are equal to or less thanabout any of 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000,2000, 3000, 4000, or 5000 μM do not substantially inhibit (e.g., inhibitby less than about any of 10, 8, 6, 4, 3, 2, 1%) the ability of thefeedback-resistant mevalonate kinase polypeptide to bind ATP, bindmevalonate, or convert mevalonate to mevalonate-5-phosphate. In someembodiments, the cells express (i) a heterologous nucleic acid encodinga second mevalonate kinase polypeptide or (ii) a duplicate copy of anucleic acid encoding a second mevalonate kinase polypeptide (such as afeedback-resistant or feedback-inhibited mevalonate kinase polypeptide).In some embodiments, the second mevalonate kinase polypeptide is aLactobacillus mevalonate kinase polypeptide (e.g., a Lactobacillus sakeimevalonate kinase polypeptide), a yeast mevalonate kinase polypeptide(e.g., a Saccharomyces cerevisia mevalonate kinase polypeptide), aStreptococcus mevalonate kinase polypeptide (e.g., a Streptococcuspneumoniae mevalonate kinase polypeptide), or a Streptomyces mevalonatekinase polypeptide (e.g., a Streptomyces CL190 mevalonate kinasepolypeptide). In some embodiments, the cells further comprise aheterologous nucleic acid or a duplicate copy of an endogenous nucleicacid encoding an isoprene synthase polypeptide. In some embodiments, thecells further comprise one or more heterologous nucleic acids or one ormore additional copies of an endogenous nucleic acid encoding anisoprene synthase polypeptide. In some embodiments, the heterologousnucleic acid encodes for an MVA pathway enzyme or a DXP pathway enzyme.In some embodiments, the MVA pathway enzyme is mevalonate kinase. Insome embodiments, the cells have a heterologous nucleic acid that (i)encodes an isoprene synthase polypeptide and (ii) is operably linked toa promoter. In some embodiments, the cells in culture produce greaterthan about 400 nmole/g_(wcm)/hr of isoprene. In some embodiments, thecells in culture convert more than about 0.002% of the carbon in a cellculture medium into isoprene.

In some embodiments of any of the cells, the cells are cultured in aculture medium that includes a carbon source, such as, but not limitedto, a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. In some embodiments, the cells are culturedunder limited glucose conditions.

In another aspect, the invention provides for one or more compositionsfor producing isoprene comprising any of the cells described herein.

In one aspect, the invention features methods of producing isoprene,such as methods of using any of the cells described herein to produceisoprene. In some embodiments, the method involves culturing cellscomprising (i) one or more non-modified nucleic acids encodingfeedback-resistant mevalonate kinase polypeptides or (ii) one or moreadditional copies of an endogenous nucleic acid encoding afeedback-resistant mevalonate kinase polypeptide. In another embodiment,the method further comprises recovering the isoprene. In someembodiments, the method involves culturing cells comprising (i) aheterologous nucleic acid encoding a feedback-resistant mevalonatekinase polypeptide (e.g., a Methanosarcina mazei mevalonate kinasepolypeptide or a homolog thereof) or (ii) a duplicate copy of anendogenous nucleic acid encoding a feedback-resistant mevalonate kinasepolypeptide (e.g., a Methanosarcina mazei mevalonate kinase polypeptideor a homolog thereof). In some embodiments, the cells are cultured undersuitable culture conditions for the production of isoprene, and isopreneis produced. In some embodiments, the mevalonate kinase nucleic acid isoperably linked to a promoter. In some embodiments, thefeedback-resistant mevalonate kinase polypeptide is an archaealmevalonate kinase polypeptide (e.g., a Methanosarcina mazei mevalonatekinase polypeptide). In some embodiments, concentrations of geranyldiphosphate (GPP) or farnesyl diphosphate (FPP) that are equal to orless than about any of 20, 30, 40, 50, 60, 70, 80, 90, or 100 μM do notsubstantially inhibit (e.g., inhibit by less than about any of 10, 8, 6,4, 3, 2, 1%) the ability of the feedback-resistant mevalonate kinasepolypeptide to bind ATP, bind mevalonate, or convert mevalonate tomevalonate-5-phosphate. In some embodiments, concentrations of3,3-dimethylallyl diphosphate (DMAPP) that are equal to or less thanabout any of 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000,2000, 3000, 4000, or 5000 μM do not substantially inhibit (e.g., inhibitby less than about any of 10, 8, 6, 4, 3, 2, 1%) the ability of thefeedback-resistant mevalonate kinase polypeptide to bind ATP, bindmevalonate, or convert mevalonate to mevalonate-5-phosphate.

The invention also provides for methods of increasing the rate or fluxof production of 3,3-dimethylallyl diphosphate (DMAPP), isopentenyldiphosphate (IPP), or a product derived from 3,3-dimethylallyldiphosphate (DMAPP) or isopentenyl diphosphate (IPP) comprising: (a)culturing cells of claim 1 wherein the cells are cultured under suitableculture conditions for increasing the rate or flux of production of3,3-dimethylallyl diphosphate (DMAPP), isopentenyl diphosphate (IPP), ora product derived from 3,3-dimethylallyl diphosphate (DMAPP) orisopentenyl diphosphate (IPP), and (b) producing 3,3-dimethylallyldiphosphate (DMAPP), isopentenyl diphosphate (IPP), or a product derivedfrom 3,3-dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate(IPP). In one embodiment, the product derived from 3,3-dimethylallyldiphosphate (DMAPP) or isopentenyl diphosphate (IPP) is isoprene.

The invention also provides for methods of producing or increasing theproduction of 3,3-dimethylallyl diphosphate (DMAPP), isopentenyldiphosphate (IPP), or a product derived from 3,3-dimethylallyldiphosphate (DMAPP) or isopentenyl diphosphate (IPP) by (a) culturingcells comprising (i) a heterologous nucleic acid encoding afeedback-resistant archaeal mevalonate kinase polypeptide or (ii) aduplicate copy of an endogenous nucleic acid encoding afeedback-resistant archaeal mevalonate kinase polypeptide, wherein thecells are cultured under suitable culture conditions for the productionof 3,3-dimethylallyl diphosphate (DMAPP), isopentenyl diphosphate (IPP),or a product derived from 3,3-dimethylallyl diphosphate (DMAPP) orisopentenyl diphosphate (IPP), and (b) producing 3,3-dimethylallyldiphosphate (DMAPP), isopentenyl diphosphate (IPP), or a product derivedfrom 3,3-dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate(IPP). In some embodiments, the product derived from 3,3-dimethylallyldiphosphate (DMAPP) or isopentenyl diphosphate (IPP) is isoprene. Inanother embodiment, the feedback-resistant archaeal mevalonate kinasepolypeptide is M. Mazei mevalonate kinase.

In some embodiments, the cells express (i) a heterologous nucleic acidencoding a second mevalonate kinase polypeptide or (ii) a duplicate copyof a nucleic acid encoding a second mevalonate kinase polypeptide (suchas a feedback-resistant or feedback-inhibited mevalonate kinasepolypeptide). In some embodiments, the second mevalonate kinasepolypeptide is a Lactobacillus mevalonate kinase polypeptide (e.g., aLactobacillus sakei mevalonate kinase polypeptide), a yeast mevalonatekinase polypeptide (e.g., a Saccharomyces cerevisiae mevalonate kinasepolypeptide), a Streptococcus mevalonate kinase polypeptide (e.g., aStreptococcus pneumoniae mevalonate kinase polypeptide), or aStreptomyces mevalonate kinase polypeptide (e.g., a Streptomyces CL190mevalonate kinase polypeptide). In some embodiments, the cells furthercomprise a heterologous nucleic acid or a duplicate copy of anendogenous nucleic acid encoding an isoprene synthase polypeptide. Insome embodiments, the cells have a heterologous nucleic acid that (i)encodes an isoprene synthase polypeptide and (ii) is operably linked toa promoter. In some embodiments, the method involves culturing cellsunder conditions sufficient to produce greater than about 400nmole/g_(wcm)/hr of isoprene. In some embodiments, the method includesculturing cells under conditions sufficient to convert more than about0.002% of the carbon (mol/mol) in a cell culture medium into isoprene.

In some embodiments of the methods, the method also includes recoveringisoprene produced by the cells. In some embodiments, the method includespurifying isoprene produced by the cells. In some embodiments, themethod includes polymerizing the isoprene. In some embodiments, thecells are cultured in a culture medium that includes a carbon source,such as, but not limited to, a carbohydrate, glycerol, glycerine,dihydroxyacetone, one-carbon source, oil, animal fat, animal oil, fattyacid, lipid, phospholipid, glycerolipid, monoglyceride, diglyceride,triglyceride, renewable carbon source, polypeptide (e.g., a microbial orplant protein or peptide), yeast extract, component from a yeastextract, or any combination of two or more of the foregoing. In someembodiments, the cells are cultured under limited glucose conditions. Invarious embodiments, the amount of isoprene produced (such as the totalamount of isoprene produced or the amount of isoprene produced per literof broth per hour per OD₆₀₀) during stationary phase is greater than orabout 2 or more times the amount of isoprene produced during the growthphase for the same length of time. In some embodiments, the gas phasecomprises greater than or about 9.5% (volume) oxygen, and theconcentration of isoprene in the gas phase is less than the lowerflammability limit or greater than the upper flammability limit. Inparticular embodiments, (i) the concentration of isoprene in the gasphase is less than the lower flammability limit or greater than theupper flammability limit, and (ii) the cells produce greater than about400 nmole/g_(wcm)/hr of isoprene.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time.

In some embodiments, at least a portion of the isoprene is in a gasphase. In some embodiments, at least a portion of the isoprene is in aliquid phase (such as a condensate). In some embodiments, at least aportion of the isoprene is in a solid phase. In some embodiments, atleast a portion of the isoprene is adsorbed to a solid support, such asa support that includes silica and/or activated carbon. In someembodiments, the composition includes ethanol. In some embodiments, thecomposition includes between about 75 to about 90% by weight of ethanol,such as between about 75 to about 80%, about 80 to about 85%, or about85 to about 90% by weight of ethanol. In some embodiments, thecomposition includes between about 4 to about 15% by weight of isoprene,such as between about 4 to about 8%, about 8 to about 12%, or about 12to about 15% by weight of isoprene.

In some embodiments, the invention also features systems that includeany of the cells and/or compositions described herein. In someembodiments, the system includes a reactor that chamber comprises cellsin culture that produce greater than about 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, or morenmole/g_(wcm)/hr isoprene. In some embodiments, the system is not aclosed system. In some embodiments, at least a portion of the isopreneis removed from the system. In some embodiments, the system includes agas phase comprising isoprene. In various embodiments, the gas phasecomprises any of the compositions described herein.

In one aspect, the invention provides a tire comprising polyisoprene. Insome embodiments, the polyisoprene is produced by (i) polymerizingisoprene in any of the compositions described herein or (ii)polymerizing isoprene recovered from any of the compositions describedherein. In some embodiments, the polyisoprene comprisescis-1,4-polyisoprene. In another aspect, the invention provides methodsof manufacturing a tire wherein the improvement comprises using any oneor more the compositions, cells, systems and/or methods described hereinto produce isoprene for the manufacture of the tire.

In some embodiments of any of the compositions, systems, and methods ofthe invention, a nonflammable concentration of isoprene in the gas phaseis produced. In some embodiments, the gas phase comprises less thanabout 9.5% (volume) oxygen. In some embodiments, the gas phase comprisesgreater than or about 9.5% (volume) oxygen, and the concentration ofisoprene in the gas phase is less than the lower flammability limit orgreater than the upper flammability limit. In some embodiments, theportion of the gas phase other than isoprene comprises between about 0%to about 100% (volume) oxygen, such as between about 10% to about 100%(volume) oxygen. In some embodiments, the portion of the gas phase otherthan isoprene comprises between about 0% to about 99% (volume) nitrogen.In some embodiments, the portion of the gas phase other than isoprenecomprises between about 1% to about 50% (volume) CO₂.

In some embodiments of any of the aspects of the invention, the cells inculture produce isoprene at greater than or about 400, 500, 600, 700,800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,or more nmole/g_(wcm)/hr isoprene. In some embodiments of any of theaspects of the invention, the cells in culture convert greater than orabout 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbonin the cell culture medium into isoprene. In some embodiments of any ofthe aspects of the invention, the cells in culture produce isoprene atgreater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500,600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000,4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram of cells forthe wet weight of the cells/hr (ng/g_(wcm)/h). In some embodiments ofany of the aspects of the invention, the cells in culture produce acumulative titer (total amount) of isoprene at greater than or about 1,10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900,1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000,50,000, 100,000, or more mg of isoprene/L of broth (mg/L_(broth),wherein the volume of broth includes the volume of the cells and thecell medium). Other exemplary rates of isoprene production and totalamounts of isoprene production are disclosed herein.

In some embodiments of any of the aspects of the invention, the cellsfurther comprise a heterologous nucleic acid encoding an IDIpolypeptide. In some embodiments of any of the aspects of the invention,the cells further comprise an insertion of a copy of an endogenousnucleic acid encoding an IDI polypeptide. In some embodiments of any ofthe aspects of the invention, the cells further comprise a heterologousnucleic acid encoding a DXS polypeptide. In some embodiments of any ofthe aspects of the invention, the cells further comprise an insertion ofa copy of an endogenous nucleic acid encoding a1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide. In someembodiments of any of the aspects of the invention, the cells furthercomprise one or more nucleic acids encoding an IDI(Isopentenyl-diphosphate delta-isomerase) polypeptide and a DXSpolypeptide. In some embodiments of any of the aspects of the invention,one nucleic acid encodes the isoprene synthase polypeptide, IDIpolypeptide, and DXS polypeptide. In some embodiments of any of theaspects of the invention, one vector encodes the isoprene synthasepolypeptide, IDI polypeptide, and DXS polypeptide. In some embodiments,the vector comprises a selective marker, such as an antibioticresistance nucleic acid.

In some embodiments of any of the aspects of the invention, the cellsexpress a second MVA pathway polypeptide (other than the mevalonatekinase polypeptide). In some embodiments, the second MVA pathwaypolypeptide is an acetyl-CoA acetyltransferase polypeptide,3-hydroxy-3-methylglutaryl-CoA synthase polypeptide,3-hydroxy-3-methylglutaryl-CoA reductase polypeptide, phosphomevalonatekinase polypeptide, diphosphomevalonate decarboxylase polypeptide, orisopentenyl-diphosphate delta-isomerase polypeptide. In someembodiments, the cells express an entire MVA pathway.

In some embodiments of any of the aspects of the invention, the cellscomprise a heterologous nucleic acid encoding an MVA pathway polypeptide(such as an MVA pathway polypeptide from Saccharomyces cerevisia orEnterococcus faecalis). In some embodiments of any of the aspects of theinvention, the cells further comprise an insertion of a copy of anendogenous nucleic acid encoding an MVA pathway polypeptide (such as anMVA pathway polypeptide from Saccharomyces cerevisia or Enterococcusfaecalis). In some embodiments of any of the aspects of the invention,the cells comprise an isoprene synthase, DXS, and MVA pathway nucleicacid. In some embodiments of any of the aspects of the invention, thecells comprise an isoprene synthase nucleic acid, a DXS nucleic acid, anIDI nucleic acid, and a MVA pathway nucleic (in addition to the IDInucleic acid).

In some embodiments of any of the aspects of the invention, the isoprenesynthase polypeptide is a polypeptide from a plant such as Pueraria(e.g., Pueraria montana or Pueraria lobata) or Populus (e.g., Populustremuloides, Populus alba, Populus nigra, Populus trichocarpa, or thehybrid, Populus alba×Populus tremula).

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

In some embodiments of any of the aspects of the invention, the cellsare bacterial cells, such as gram-positive bacterial cells (e.g.,Bacillus cells such as Bacillus subtilis cells or Streptomyces cellssuch as Streptomyces lividans, Streptomyces coelicolor, or Streptomycesgriseus cells). In some embodiments of any of the aspects of theinvention, the cells are gram-negative bacterial cells (e.g.,Escherichia cells such as Escherichia coli cells or Pantoea cells suchas Pantoea citrea cells). In some embodiments of any of the aspects ofthe invention, the cells are fungal, cells such as filamentous fungalcells (e.g., Trichoderma cells such as Trichoderma reesei cells orAspergillus cells such as Aspergillus oryzae and Aspergillus niger) oryeast cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells).

In some embodiments of any of the aspects of the invention, themicrobial polypeptide carbon source includes one or more polypeptidesfrom yeast or bacteria. In some embodiments of any of the aspects of theinvention, the plant polypeptide carbon source includes one or morepolypeptides from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, or linseed.

In one aspect, the invention features a product produced by any of thecompositions or methods of the invention. It is to be understood thatone, some, or all of the properties of the various embodiments describedherein may be combined to form other embodiments of the presentinvention.

All publications, patents and patent applications referenced in thisspecification are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase genecodon-optimized for expression in E. coli (SEQ ID NO:1). The atg startcodon is in italics, the stop codon is in bold and the added PstI siteis underlined.

FIG. 2 is a map of pTrcKudzu.

FIGS. 3A-3C are the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). TheRBS is underlined, the kudzu isoprene synthase start codon is in boldcapitol letters and the stop codon is in bold, capitol, italics letters.The vector backbone is pTrcHis2B.

FIG. 4 is a map of pETNHisKudzu.

FIGS. 5A-5C are the nucleotide sequence of pETNHisKudzu (SEQ ID NO:5).

FIG. 6 is a map of pCL-lac-Kudzu.

FIGS. 7A-7C are the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:7).

FIG. 8A is a graph showing the production of isoprene in E. coli BL21cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21cells with pETNHisKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coliBL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentationof E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoacitrea. Control cells without recombinant kudzu isoprene synthase. Greydiamonds represent isoprene synthesis, black squares represent OD₆₀₀.

FIG. 10B is a graph showing the production of isoprene in Panteoa citreaexpressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis,black squares represent OD₆₀₀.

FIG. 10C is a graph showing the production of isoprene in Panteoa citreaexpressing pTrcKudzu. Grey diamonds represent isoprene synthesis, blacksquares represent OD₆₀₀.

FIG. 11 is a graph showing the production of isoprene in Bacillussubtilis expressing recombinant isoprene synthase. BG3594comK is a B.subtilis strain without plasmid (native isoprene production).CF443-BG3594comK is a B. subtilis strain with pBSKudzu (recombinantisoprene production). IS on the y-axis indicates isoprene.

FIGS. 12A-12C are the nucleotide sequence of pBS Kudzu #2 (SEQ IDNO:57).

FIG. 13 is the nucleotide sequence of kudzu isoprene synthasecodon-optimized for expression in Yarrowia (SEQ ID NO:8).

FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase genecodon-optimized for expression in Yarrowia.

FIGS. 15A-15C are the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQID NO:11).

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Puerariamontana) isoprene gene codon-optimized for expression in Yarrowia (SEQID NO:12).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar(Populus alba×Populus tremula) isoprene synthase gene (SEQ ID NO:13).The ATG start codon is in bold and the stop codon is underlined.

FIG. 18A shows a schematic outlining construction of vectors pYLA 1,pYL1 and pYL2 (SEQ ID NO:73-77, SEQ ID NO:79).

FIG. 18B shows a schematic outlining construction of the vectorpYLA(POP1) (SEQ ID NO:72-73).

FIG. 18C shows a schematic outlining construction of the vectorpYLA(KZ1) (SEQ ID NO:70-71).

FIG. 18D shows a schematic outlining construction of the vectorpYL-I(KZ1)

FIG. 18E shows a schematic outlining construction of the vectorpYLI(MAP29)

FIG. 18F shows a schematic outlining construction of the vectorpYLA(MAP29)

FIG. 19A shows the MVA and DXP metabolic pathways for isoprene (based onF. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). Thefollowing description includes alternative names for each polypeptide inthe pathways and a reference that discloses an assay for measuring theactivity of the indicated polypeptide (each of these references are eachhereby incorporated by reference in their entireties, particularly withrespect to assays for polypeptide activity for polypeptides in the MVAand DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoAacetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR;3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J.Bacteriol., 184: 2116-2122, 2002; MVK or MK; Mevalonate kinase, ERG12,EC 2.7.1.36. Assay: Curr Genet 19:9-14, 1991. PMK; Phosphomevalonatekinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991;DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay:Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphatedelta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem.264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphatesynthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR;1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay:Eur. J. Biochem. 269:4446-4457, 2002; MCT;4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60.Assay: PNAS, 97: 6451-6456, 2000; CMK;4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148.Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS,96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem.,70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphatereductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 19B illustrates the classical and modified MVA pathways. 1,acetyl-CoA acetyltransferase (AACT); 2, HMG-CoA synthase (HMGS); 3,HMG-CoA reductase (HMGR); 4, mevalonate kinase (MVK); 5,phosphomevalonate kinase (PMK); 6, diphosphomevalonate decarboxylase(MVD or DPMDC); 7, isopentenyl diphosphate isomerase (IDI); 8,phosphomevalonate decarboxylase (PMDC); 9, isopentenyl phosphate kinase(IPK). The classical MVA pathway proceeds from reaction 1 throughreaction 7 via reactions 5 and 6, while a modified MVA pathway goesthrough reactions 8 and 9. P and PP in the structural formula arephosphate and pyrophosphate, respectively. This figure was taken fromKoga and Morii, Microbiology and Mol. Biology Reviews, 71:97-120, 2007,which is incorporated by reference in its entirety, particularly withrespect to nucleic acids and polypeptides of the modified MVA pathway.The modified MVA pathway is present, for example, in some archaealorganisms, such as Methanosarcina mazei.

FIG. 20 shows graphs representing results of the GC-MS analysis ofisoprene production by recombinant Y. lipolytica strains without (left)or with (right) a kudzu isoprene synthase gene. The arrows indicate theelution time of the authentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIGS. 22A-22D are the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQID NO:20).

FIG. 23A is a graph showing production of isoprene from glucose inBL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol).The x-axis is time after induction; the y-axis is OD₆₀₀ and the y2-axisis total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose inBL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG(400 μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ andthe y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Diamonds represent OD₆₀₀,circles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD₆₀₀ and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD₆₀₀, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23H is a graph showing production of isoprene from glucose inBL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction withIPTG (400 μmol). The x-axis is time after inoculation; the y-axis isOD600 and the y2-axis is total productivity of isoprene (μg/L headspaceor specific productivity (μg/L headspace/OD). Diamonds represent OD600,triangles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIGS. 25A-25D are the nucleotide sequence of pTrcKKDyIkIS kan (SEQ IDNO:33).

FIG. 26 is a map of pCL PtrcUpperPathway.

FIGS. 27A-27D are the nucleotide sequence of pCL PtrcUpperPathway (SEQID NO:46).

FIG. 28 shows a map of the cassette containing the lower MVA pathway andyeast idi for integration into the B. subtilis chromosome at the nprElocus. nprE upstream/downstream indicates 1 kb each of sequence from thenprE locus for integration. aprE promoter (alkaline serine proteasepromoter) indicates the promoter (−35, −10, +1 transcription start site,RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene.RBS-PMK indicates the yeast phosphomevalonate kinase gene with aBacillus RBS upstream of the start site. RBS-MPD indicates the yeastdiphosphomevalonate decarboxylase gene with a Bacillus RBS upstream ofthe start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBSupstream of the start site. Terminator indicates the terminator alkalineserine protease transcription terminator from B. amyliquefaciens. SpecRindicates the spectinomycin resistance marker. “nprE upstream repeat foramp.” indicates a direct repeat of the upstream region used foramplification.

FIGS. 29A-29D are the nucleotide sequence of cassette containing thelower MVA pathway and yeast idi for integration into the B. subtilischromosome at the nprE locus (SEQ ID NO:47).

FIG. 30 is a map of p9796-poplar.

FIGS. 31A and 31B are the nucleotide sequence of p9796-poplar (SEQ IDNO:48).

FIG. 32 is a map of pTrcPoplar.

FIGS. 33A-33C are the nucleotide sequence of pTrcPoplar (SEQ ID NO:49).

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIGS. 35A-35C are the nucleotide sequence of pTrcKudzu yIDI Kan (SEQ IDNO:50).

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIGS. 37A-37C are the nucleotide sequence of pTrcKudzuDXS Kan (SEQ IDNO:51).

FIG. 38 is a map of pCL PtrcKudzu.

FIGS. 39A-39C are the nucleotide sequence of pCL PtrcKudzu (SEQ IDNO:52).

FIG. 40 is a map of pCL PtrcKudzu A3.

FIGS. 41A-41C are the nucleotide sequence of pCL PtrcKudzu A3 (SEQ IDNO:53).

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIGS. 43A-43C are the nucleotide sequence of pCL PtrcKudzu yIDI (SEQ IDNO:54).

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIGS. 45A-45D are the nucleotide sequence of pCL PtrcKudzu DXS (SEQ IDNO:55).

FIG. 46A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 46B and 46C are the nucleotide sequence of the M. mazei archaeallower Pathway operon (SEQ ID NO:102).

FIG. 47A is a map of MCM382-pTrcKudzuMVK(mazei).

FIGS. 47B and 47C are the nucleotide sequence ofMCM382-pTrcKudzuMVK(mazei) (SEQ ID NO:103).

FIGS. 48A-48C are graphs demonstrating the effect of yeast extract ofisoprene production. FIG. 48A is the time course of optical densitywithin fermentors fed with varying amounts of yeast extract. FIG. 48B isthe time course of isoprene titer within fermentors fed with varyingamounts of yeast extract. The titer is defined as the amount of isopreneproduced per liter of fermentation broth. FIG. 48C shows the effect ofyeast extract on isoprene production in E. coli grown in fed-batchculture.

FIG. 49 shows graphs demonstrating isoprene production from a 500 Lbioreactor with E. coli cells containing the pTrcKudzu+yIDI+DXS plasmid.Panel A shows the time course of optical density within the 500-Lbioreactor fed with glucose and yeast extract. Panel B shows the timecourse of isoprene titer within the 500-L bioreactor fed with glucoseand yeast extract. The titer is defined as the amount of isopreneproduced per liter of fermentation broth. Panel C shows the time courseof total isoprene produced from the 500-L bioreactor fed with glucoseand yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIGS. 51A-51C are the nucleotide sequence of pJMupperpathway2 (SEQ IDNO:56).

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillusexpressing recombinant kudzu isoprene synthase in 14 liter fed batchfermentation. Black diamonds represent a control strain (BG3594comK)without recombinant isoprene synthase (native isoprene production) andgrey triangles represent Bacillus with pBSKudzu (recombinant isopreneproduction).

FIG. 53B is a graph showing isoprene production during fermentation timeof Bacillus expressing recombinant kudzu isoprene synthase in 14 literfed batch fermentation. Black diamonds represent a control strain(BG3594comK) without recombinant isoprene synthase (native isopreneproduction) and grey triangles represent Bacillus with pBSKudzu(recombinant isoprene production).

FIG. 54 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 55 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 56 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 57A is a map of MCM376-MVK from M. mazei archaeal Lowerin pET200D.

FIGS. 57B and 57C are the nucleotide sequence of MCM376-MVK from M.mazei archaeal Lowerin pET200D (SEQ ID NO:104).

FIGS. 58A-58D are graphs showing the kinetics of yeast and M. mazeimevalonate kinases. FIG. 58A is a graph of the rate vs. [ATP] for yeastmevalonate kinase. FIG. 58B is a graph of the rate vs. [mevalonate] foryeast mevalonate kinase. FIG. 58C is a graph of the rate vs. [ATP] forM. mazei mevalonate kinase. FIG. 58D is a graph of the rate vs.[mevalonate] for M. mazei mevalonate kinase.

FIG. 59A is a map of MCM 383-pTrcKudzuMVK (S. cerevisiae).

FIGS. 59B and 59C are the nucleotide sequence of MCM 383-pTrcKudzuMVK(S. cerevisiae) (SEQ ID NO:105).

FIGS. 60A-60C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 150-L bioreactor fed withglucose.

FIGS. 61A-61C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIGS. 62A-62C are the time courses of optical density, mevalonic acidtiter, and specific productivity within the 15-L bioreactor fed withglucose.

FIGS. 63A-63C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 64A-64C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 65A-65C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 66A-66C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIGS. 67A-67C are the time courses of optical density, isoprene titer,and specific productivity within the 15-L bioreactor fed with glucose.

FIG. 68 is a graph of the calculated adiabatic flame temperatures forSeries A as a function of fuel concentration for various oxygen levels.The figure legend lists the curves in the order in which they appear inthe graph. For example, the first entry in the figure legend (isoprenein air at 40° C.) corresponds to the highest curve in the graph.

FIG. 69 is a graph of the calculated adiabatic flame temperatures forSeries B as a function of fuel concentration for various oxygen levelswith 4% water. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 70 is a graph of the calculated adiabatic flame temperatures forSeries C as a function of fuel concentration for various oxygen levelswith 5% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 71 is a graph of the calculated adiabatic flame temperatures forSeries D as a function of fuel concentration for various oxygen levelswith 10% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 72 is a graph of the calculated adiabatic flame temperatures forSeries E as a function of fuel concentration for various oxygen levelswith 15% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 73 is a graph of the calculated adiabatic flame temperatures forSeries F as a function of fuel concentration for various oxygen levelswith 20% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 74 is a graph of the calculated adiabatic flame temperatures forSeries G as a function of fuel concentration for various oxygen levelswith 30% CO₂. The figure legend lists the curves in the order in whichthey appear in the graph.

FIG. 75A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series A.

FIG. 75B is a graph of the flammability results from the CAFT model forSeries A in FIG. 68 plotted as volume percent.

FIG. 76A is a table of the conversion of the CAFT Model results fromweight percent to volume percent for series B.

FIG. 76B is a graph of the flammability results from the CAFT model forSeries B in FIG. 69 plotted as volume percent.

FIG. 77 is a figure of the flammability test vessel.

FIG. 78A is a graph of the flammability Curve for Test Series 1: 0%Steam, 0 psig, and 40° C.

FIG. 78B is a table summarizing the explosion and non-explosion datapoints for Test Series 1.

FIG. 78C is a graph of the flammability curve for Test Series 1 comparedwith the CAFT Model.

FIG. 79A is a graph of the flammability curve for Test Series 2: 4%Steam, 0 psig, and 40° C.

FIG. 79B is a table summarizing the explosion and non-explosion datapoints for Test Series 2.

FIG. 79C is a graph of the flammability curve for Test Series 2 comparedwith the CAFT Model.

FIGS. 80A and 80B are a table of the detailed experimental conditionsand results for Test Series 1.

FIG. 81 is a table of the detailed experimental conditions and resultsfor Test Series 2.

FIG. 82 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 3 atmospheres of pressure.

FIG. 83 is a graph of the calculated adiabatic flame temperature plottedas a function of fuel concentration for various nitrogen/oxygen ratiosat 1 atmosphere of pressure.

FIG. 84 is a graph of the flammability envelope constructed using datafrom FIG. 82 and following the methodology described in Example 21. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 85 is a graph of the flammability envelope constructed using datafrom FIG. 83 and following the methodology described in Example 21. Theexperimental data points (circles) are from tests described herein thatwere conducted at 1 atmosphere initial system pressure.

FIG. 86A is a GC/MS chromatogram of fermentation off-gas.

FIG. 86B is an expansion of FIG. 86A to show minor volatiles present infermentation off-gas.

FIG. 87A is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −78° C.

FIG. 87B is a GC/MS chromatogram of trace volatiles present in off-gasfollowing cryo-trapping at −196° C.

FIG. 87C is an expansion of FIG. 87B.

FIG. 87D is an expansion of FIG. 87C.

FIGS. 88A and 88B are GC/MS chromatogram comparing C5 hydrocarbons frompetroleum-derived isoprene (FIG. 88A) and biologically produced isoprene(FIG. 88B). The standard contains three C5 hydrocarbon impuritieseluting around the main isoprene peak (FIG. 88A). In contrast,biologically produced isoprene contains amounts of ethanol and acetone(run time of 3.41 minutes) (FIG. 88A).

FIG. 89 is a graph of the analysis of fermentation off-gas of an E. coliBL21 (DE3) pTrcIS strain expressing a Kudzu isoprene synthase and fedglucose with 3 g/L yeast extract.

FIG. 90 shows the structures of several impurities that are structurallysimilar to isoprene and may also act as polymerization catalyst poisons.

FIG. 91 is a map of pTrcHis2AUpperPathway (also called pTrcUpperMVA).

FIGS. 92A-92C are the nucleotide sequence of pTrcHis2AUpperPathway (alsocalled pTrcUpperMVA) (SEQ ID NO:86).

FIG. 93 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 94 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 95 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIGS. 96A and 96B are Lineweaver-Burke plots for DMAPP inhibition ofyeast mevalonate kinase. FIG. 96A shows that DMAPP displays competitiveinhibition with respect to ATP. FIG. 96B shows that DMAPP displaysuncompetitive inhibition with respect to mevalonate.

FIGS. 97A-97C are graphs showing the inhibition of yeast mevalonatekinase with respect to ATP. FIG. 97A shows the percent of activitywithout inhibitor vs. [DMAPP] at [ATP] equal to K_(MappATP). FIG. 97Bshows the percent of activity without inhibitor vs. [GPP] at [ATP] equalto K_(MappATP). FIG. 97C shows the percent of activity without inhibitorvs. [FPP] at [ATP] equal to K_(MappATP).

FIGS. 98A-98C are graphs showing the inhibition of yeast mevalonatekinase with respect to mevalonate. FIG. 98A shows the percent ofactivity without inhibitor vs. [DMAPP] at [mevalonate] equal toK_(MappMev). FIG. 98B shows the percent of activity without inhibitorvs. [GPP] at [mevalonate] equal to K_(MappMev). FIG. 98C shows thepercent of activity without inhibitor vs. [FPP] at [mevalonate] equal toK_(MappMev).

FIG. 99 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 100 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 101 is a time course of isoprene specific activity from the 15-Lbioreactor fed with glucose.

FIG. 102 is a map of pCLPtrcUpperPathwayHGS2 (also referred to as pCLUpperHGS2).

FIGS. 103A-103C are the nucleotide sequence of pCLPtrcUpperPathwayHGS2(SEQ ID NO:87).

FIG. 104 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 105 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 106 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 107 is a map of plasmid MCM330.

FIGS. 108A-108C are the nucleotide sequence of plasmid MCM330 (SEQ IDNO:90).

FIG. 109 is a map of pET24D-Kudzu.

FIGS. 110A and 110B are the nucleotide sequence of pET24D-Kudzu (SEQ IDNO:101).

FIG. 111A is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 111B is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 111C is a time course of specific productivity of isoprene in the15-L bioreactor fed with glucose.

FIG. 112A is a graph of the growth of MCM127 in TM3 media at 30° C.measured as optical density (OD600). One culture was induced with 150 μMIPTG 4 hours after inoculation.

FIG. 112B is a graph of the accumulated key metabolic intermediatesafter induction of MCM127 with 150 μM IPTG. The culture was induced 4hours after inoculation and samples were analyzed using LCMS.

FIGS. 112C-112K are isoprene fermentation expressing genes from the MVApathway and grown in fed-batch culture at the 15-L scale in different E.coli strains (MCM343 strain (FIGS. 112C-112E); MCM127 strain (FIGS.112F-112H); dxr knock-out strain (FIGS. 112I-112K)). FIGS. 112C, 112F,and 112I show the time course of optical density within the 15-Lbioreactor fed with glucose in MCM343 strain, MCM127 strain, and dxrknock-out strain, respectively. FIGS. 112D, 112G, and 112J are the timecourse of isoprene titer within the 15-L bioreactor fed with glucose inMCM343 strain, MCM127 strain, and dxr knock-out strain, respectively.The titer is defined as the amount of isoprene produced per liter offermentation broth. FIGS. 112E, 112H, and 112K are the time course oftotal isoprene produced from the 15-L bioreactor fed with glucose inMCM343 strain, MCM127 strain, and dxr knock-out strain, respectively.

FIGS. 112L-112N depict the construction and phenotype of the dxr mutantin E. coli. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (dxr) wasdeleted using the GeneBridges Quick & Easy E. coli Gene Deletion Kit.FIG. 112L shows the chromosomal location of dxr (from EcoCyc) and theapproximate primer binding sites for testing the insertion of the GBresistance cassette. FIG. 112M is a PCR analysis of dxr deletion strains(in MG1655) using primers dxrTest1 and GBprimer2 (GB2), and dxrTest2 andGBprimerDW (GB3). PCR products were run on an Egel (Invitrogen)according to the manufacturer's protocol. FIG. 112N shows the inhibitionof the growth of dxr deletion strains at 10 mM MVA. DW28 were grownovernight at 37° C. on LB medium plates containing spectinomycin 50μg/ml, chloramphenicol 25 μg/ml, and the indicated concentrations ofMVA.

FIG. 112O lists forward and reverse primers for pCL Ptrc(minus lacO)UpperPathway: forward primer MCM63 (SEQ ID NO:123) and reverse primerMCM64 (SEQ ID NO:124).

FIG. 112P is a map of MCM184-pCL Ptrc(minus lacO) UpperPathway.

FIG. 112Q-112S are the nucleotide sequence of MCM184 (SEQ ID NO:125).

FIG. 112T lists PCR and sequencing primers for pCL Ptrc (ΔlacO)KKDyI:primer EL-976 (SEQ ID NO:126), primer EL-977 (SEQ ID NO:127), and primerEL-978 (SEQ ID NO:128).

FIG. 112U is a map of pCL Ptrc (ΔlacO)KKDyI.

FIGS. 112V-112X are the nucleotide sequence of pCL Ptrc (ΔlacO)KKDyI(SEQ ID NO:129).

FIGS. 113A-113D demonstrate that over-expression of MVK and isoprenesynthase results in increased isoprene production. Accumulated isopreneand CO₂ from MCM401 and MCM343 during growth on glucose in 100 mLbioreactors with 100 and 200 uM IPTG induction of isoprene productionwas measured over a 22 hour time course. FIG. 113A is a graph of theaccumulated isoprene (%) from MCM343. FIG. 113B is a graph of theaccumulated isoprene (%) from MCM401. FIG. 113C is a graph of theaccumulated CO₂(%) from MCM343. FIG. 113D is a graph of the accumulatedCO₂(%) from MCM401.

FIG. 114 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 115 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 116 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 117 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 118 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 119A is a map of plasmid MCM94-pTrcHis2B kan.

FIGS. 119B and 119C are the nucleotide sequence of plasmidMCM94-pTrcHis2B kan (SEQ ID NO: 107).

FIG. 120 is a graph showing that over-expression of both isoprenesynthase and MVK results in an increased specific productivity ofisoprene compared to over-expression of each of the enzymes alone, orlow expression of both enzymes. The specific productivity of isopreneusing MCM343, MCM401, MCM437, and MCM438 during growth on glucose inmini-fermentations with 200 μM IPTG induction was measured over time.Error bars represent one standard deviation.

FIG. 121 is a typical elution profile of phosphorylated intermediates inthe isoprenoid pathway extracted from the MCM391 strain of E. coli after50 hours of fermentation and detected using LC-ESI-MS/MS.

FIGS. 122A-122F are graphs showing the accumulation of isoprenoidpathway intermediates in MCM401 strain of E. coli containing MVK from M.mazei upon different levels of enzyme expression. FIGS. 122A-122C showODs and specific isoprene production of the cultures grown in 14-Lfermentors, and FIGS. 122D-122F show intracellular levels of isoprenoidmetabolites. Arrows on top of the figures indicate the time points whenIPTG was added to fermentors (1-4×50 μM; 2-2×100 μM and 3-1×200 μM).

FIG. 123 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 124 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 125 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIGS. 126A and 126B are the nucleotide sequence of pDU-5 MVK from S.cerevsiae in pET-16b (SEQ ID NO:108).

FIGS. 127A and 127B are graphs showing the accumulation of isoprenoidpathway intermediates in the MCM402 strain of E. coli containing MVKfrom yeast and grown in 14-L fermentors. Arrows on the top figureindicate the time points when 50 μM IPTG doses were added to fermentors.

FIGS. 128A and 128B are graphs showing the accumulation of isoprenoidpathway intermediates in the MCM343 strain of E. coli. Arrows on the topfigure indicate the time point when 100 μM IPTG dose was added to thefermentor.

FIG. 129 is a graph of growth curves for cultures of BL21 expressingMVK, circles; MVK+PMV, triangles; MVK+PMV+MDD, squares. Cultures wereeither fed 5.8 mM MVA, filled symbols, or grown without addition of MVA,open symbols. Y-axis is OD₆₀₀. Samples were taken for analysis at timesindicated by the arrow. Numbers above the arrows correspond to E. coliBL21 cells bearing pTrcK, representing a plasmid expressing MVK (#5),pTrcKK representing a plasmid expressing MVK plus PMK (#7), and pTrcKKD,representing a plasmid expressing MVK plus PMK plus MDD (#6) were grown.

FIG. 130 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 131 is a time course of isoprene titer within the 15-L bioreactorfed with glucose. The titer is defined as the amount of isopreneproduced per liter of fermentation broth.

FIG. 132 is a time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 133 is a time course of volumetric productivity within the 15-Lbioreactor fed with glucose. The volumetric productivity is defined asthe amount of isoprene produced per liter of broth per hour.

FIG. 134 is a time course of instantaneous yield within the 15-Lbioreactor fed with glucose. The instantaneous yield is defined as theamount of isoprene (gram) produced per amount of glucose (gram) fed tothe bioreactor (w/w) during the time interval between the data points.

FIG. 135 is a time course of optical density within the 15-L bioreactorfed with glucose.

FIG. 136 is a graph of isoprene synthase (IS) activity versus volumetricproductivity in strains MCM127, MCM343, and MCM401.

FIG. 137 is an alignment of the protein sequences for mevalonate kinasesfrom Homo sapiens, Methanosarcina mazei, Streptococcus pneumoniae, andMethanococcus jannaschii (SEQ ID NOs:109-112, respectively). Alignmentswere generated using Vector Nti (Invitrogen). Light grey areahighlighted amino acids are identical; dark grey highlighted amino acidsare conserved in some of the mevalonate kinases. The boxed arearepresents the loop that is present in the mevalonate kinases from Homosapiens and M. jannaschii (sensitive to feedback inhibition) but absentin M. mazei and S. pneumoniae (feedback inhibition resistant).

FIG. 138 is M. mazei mevalonate kinase BLAST search distance tree.Exemplary potential feedback resistant mevalonate kinase polypeptidesare circled.

FIGS. 139A-139C are graphs of a model of M. mazei mevalonate kinase.FIG. 139A is a model of the M. mazei mevalonate kinase generated usingPyMol based on the Streptococcus pneumoniae (pdb 2oi2) as the startingstructure. FIG. 139B is a pictorial view of the active sites of the M.mazei mevalonate kinase, with Lys9, His16, Ser95, Ser135, Asp138, andThr174 shown as sticks. FIG. 139C shows the conserved ATP binding motifof M. mazei mevalonate kinase, containing residues ⁸⁷PVGSGKGSSAA⁹⁷.

FIGS. 140A-140G are graphs showing the change in optical density (OD)over time correlating with diphosphomevalonate inhibition of S.pneumoniae mevalonate kinase (FIGS. 140A-140B), yeast mevalonate kinase(FIGS. 140C-140D), and M. mazei mevalonate kinase (FIGS. 140E-140G).FIGS. 140A, 140C, 140E, and 140F represent reactions A (open circle), B(open square), and C (open triangle) containing mevalonate kinase in 100μM Tris and reactions E (open diamond), F (closed circle), and G (closedsquare) containing mevalonate kinase and phosphomevalonate kinase. FIGS.140B, 140D, and 140G represent reactions A (open circle), B (opensquare), and C (open triangle) containing mevalonate kinase to whichphosphomevalonate kinase has been added after the mevalonate kinasereaction is complete and reactions E (open diamond), F (closed circle),and G (closed square) containing mevalonate kinase and phosphomevalonatekinase.

FIG. 141 is a graph showing the change in optical density over timecorrelating with isopentenyl monophosphate inhibition of M. mazeimevalonate kinase. Reaction A (open circle) contains M. mazei mevalonatekinase and all necessary cofactors. Reaction B (open square) contains M.mazei mevalonate kinase, all necessary cofactors, and 100 μM isopentenylmonophosphate.

FIG. 142 is a map of pDW02 in pET200D MVK (S. pneumoniae).

FIGS. 143A and 143B are the nucleotide sequence of pDW02 in pET200D MVK(S. pneumoniae) (SEQ ID NO:106).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides for, inter alia, compositionsand methods for producing and/or increasing the production of isopreneby utilizing a feedback resistant mevalonate kinase as part of theproduction process. Mevalonate kinase (MVK or MK) polypeptidesphosphorylate mevalonate (MVA) to form mevalonate-5-phosphate (MVAP), aspart of the MVA pathway for the biosynthesis of isoprene (FIGS. 19A and19B). As used herein, the term “isoprene” or “2-methyl-1,3-butadiene”(CAS #78-79-5) refers to the direct and final volatile C5 hydrocarbonproduct from the elimination of pyrophosphate from 3,3-dimethylallylpyrophosphate (DMAPP), and does not involve the linking orpolymerization of one or more isopentenyl diphosphate (IPP) molecules toone or more DMAPP molecules.

The present invention is based in part on the surprising discovery thatthe MVK polypeptide from the archaeon Methanosarcina mazei is resistantto feedback inhibition. For example, this result is surprising in viewof a prior report that the MVK from the archaeon Methanococcusjannaschii is feedback inhibited by GPP (geranyl pyrophosphate) and FPP(farnesyl pyrophosphate) (see, for example, Huang et al., ProteinExpression and Purification 17(1):33-40, 1999; which is herebyincorporated by reference in its entirety, particularly with respect tofeedback-inhibited MVK polypeptides). Additionally, the human MVKpolypeptide is reported to be inhibited by GPP, FPP, and geranylgeranyldiphosphate (GGPP) with K_(i) values in the nanomolar range, and MVKpolypeptides from some bacterial sources are reported to be inhibited byFPP with K_(i) values in the μM range.

As used herein, the term “enzyme inhibition” denotes the inhibition ofenzymatic activity of an MVK polypeptide. Exemplary types of enzymeinhibition include substrate inhibition and feedback inhibition. Invarious embodiments, an enzymatic activity of a MVK polypeptide (such asthe conversion of MVA to MVAP) is inhibited by less than about any of90, 80, 70, 60, 50, 40, 30, 20, 10, or 5% by a compound (such as MVA)produced by the cells compared to the enzymatic activity of the MVKpolypeptide in the absence of the compound.

As used herein, the term “substrate inhibition” refers to the inhibitionof enzymatic activity of a mevalonate kinase polypeptide by thesubstrate MVA.

As used herein, the term “feedback inhibition” denotes the inhibition ofenzymatic activity of a MVK polypeptide by a metabolite downstream ofmevalonate in isoprenoid or isoprenoid biosynthesis. Metabolitesdownstream of mevalonate (MVA) in isoprenoid or isoprenoid biosynthesisinclude, but are not limited to, mevalonate-5-phosphate (MVAP),mevalonate-5-diphosphate (MVAPP), isopentenyl diphosphate (IPP), 3,3dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyldiphosphate (FPP), geranylgeranyl diphosphate (GGPP), farnesol, dolicholphosphate, phytyl-pyrophosphate, diphosphomevalonate, and isopentenylmonophosphate (IP). While not intending to be bound by any particulartheory, it is believed that feedback inhibition of MVK polypeptides isbased on allosteric regulation of MVK polypeptides by binding to the MVKpolypeptides of a metabolite downstream of mevalonate in isoprenoidbiosynthesis (see, for example, WO/2004/111214 which is herebyincorporated by reference in its entirety, particularly with respect tofeedback-inhibited and feedback-resistant MVK polypeptides).

Feedback inhibition of an MVK polypeptide can be analyzed using anystandard method known to one skilled in the art. For example, MVKpolypeptide feedback inhibition by the mevalonate downstream metabolitediphosphomevalonate can be measured using a two enzyme system employingthe MVK polypeptide in question and PMK (see, e.g., Andreassi et al.,Biochemistry, 43:16461-66, 2004, which is incorporated by reference inits entirety). In some embodiments, the feedback inhibition of an MVKpolypeptide by a potential feedback inhibitor molecule (e.g., ametabolite downstream of mevalonate in isoprenoid or isoprenoidbiosynthesis) is determined as described in Example 8, part IX (v).

By “feedback resistance” or “feedback-resistant” is meant any resistanceto feedback inhibition. Feedback resistance can be analyzed using anystandard method known to one skilled in the art. For example, MVKpolypeptide activity can be measured in two different methods. In thefirst method, ATP (or another phosphate donor) concentration is around anon-saturating concentration (i.e., a concentration around which thereaction rate is sensitive to changes of these substrate concentrations(e.g., at concentrations around the respective Km values of the MVKpolypeptide under investigation for these substrates concentration, seefor example, WO/2004/111214, which is hereby incorporated by referencein its entirety, particularly with respect to feedback-inhibited andfeedback-resistant MVK polypeptides)) and mevalonate (or mevalonateanalogue) is around a saturating concentration. In the second method,ATP (or another phosphate donor) concentration is around a saturatingconcentration and mevalonate is around a non-saturating concentration(e.g., Km values). In some embodiments, the K_(i) value for a potentialfeedback inhibitor molecule (e.g., a metabolite downstream of mevalonatein isoprenoid or isoprenoid biosynthesis) is determined as described inExample 8, part IX (i-iv).

In some embodiments in which a MVK polypeptide is feedback-resistant,concentrations of GPP or FPP that are equal to or less than about any of20, 30, 40, 50, 60, 70, 80, 90, or 100 μM do not substantially inhibit(e.g., inhibit by less than about any of 10, 8, 6, 4, 3, 2, 1%) one ormore of the following activities of a MVK polypeptide: the binding ofATP, the binding of mevalonate, and/or the conversion of MVA to MVAP. Insome embodiments, concentrations of DMAPP that are equal to or less thanabout any of 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000,2000, 3000, 4000, or 5000 μM do not substantially inhibit (e.g., inhibitby less than about any of 10, 8, 6, 4, 3, 2, 1%) one or more of thefollowing activities of a MVK polypeptide: the binding of ATP, thebinding of mevalonate, and/or the conversion of MVA to MVAP. In someembodiments, the K_(i) value of a feedback-resistant MVK polypeptide forGPP or FPP is equal to or greater than about any of 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, or 300 μM. In someembodiments, the K_(i) value of a feedback-resistant MVK polypeptide forDMAPP is equal to or greater than about any of 0.8, 1.0, 2.0, 3.0, 4.0,5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 mM.

The use of a feedback-resistant MVK polypeptide for the production ofisoprene is desirable because it results in increased MVK polypeptideactivity, and thereby reduces the accumulation of MVA and increases thesupply of MVAP for conversion to isoprene using the MVA pathway. Example8 compares the ability of DMAPP, GPP, FPP, and GGPP to inhibit MVKpolypeptides from the yeast Sacharaomyces cerevisiae and the archaeonMethanosarcina mazei. The conversion of mevalonate to phosphomevalonatecatalyzed by the M. mazei MVK polypeptide was not inhibited by DMAPP,GPP, or FPP at concentrations up to 100 μM under the conditions tested,correlating to a K_(i)>200 μM. Additionally, the M. mazei MVKpolypeptide was not inhibited by up to 5 mM DMAPP under the conditionstested. In contrast, DMAPP, GPP, and FPP are competitive inhibitors withrespect to ATP for the S. cerevisiae MVK polypeptide with inhibitionconstants (K_(i)s) of 33.2 μM, 153.3 nM, and 138.5 nM, respectively. Inaddition, DMAPP, GPP, and FPP are uncompetitive inhibitors with respectto mevalonate for the S. cerevisiae MVK polypeptide with K_(i)s of 394.6μM 2.54 μM, and 2.98 μM, respectively.

Based on the discovery that the MVK polypeptide from the archaeon M.mazei is resistant to feedback inhibition, other MVK polypeptides (suchas other archaeal MVK polypeptides) that are homologous to the M. mazeiMVK polypeptide are predicted to be feedback-resistant as well. Inparticular, sequence alignments indicate that the M. mazei MVKpolypeptide is missing a loop that is present in MVK polypeptides thatare sensitive to feedback inhibition by downstream products such as FPP(e.g., MVK polypeptides from Homo sapiens, rat, and Methanococcusjannaschii). These MVK polypeptides from H. sapiens, rat, and M.jannaschii contain a loop that is absent in MVK polypeptides that areresistant to feedback inhibition (Fu et al., Biochemistry47(12):3715-24, 2008; see, for example, FIG. 3; which is herebyincorporated by reference in its entirety, particularly with respect tofeedback-inhibited and feedback-resistant MVK polypeptides). Further,modeling studies of the M. mazei MVK suggest that it folds in a similarway in comparison to S. pneumoniae MVK, including the active site andthe ATP binding motif (Andreassi et al., Protein Sci 16(5): 983-989,2007). The human MVK contains two additional loops (residues 59-85 and93-121) that are not found in the M. mazei or S. pneumoniae MVKs. It hasbeen shown that residues found in those loops contribute to feedbackinhibition by farnesyl diphosphate (see supra Fu et al.). FIGS.139A-139C show a model of M. mazei mevalonate kinase, its active site,and its conserved ATP binding motif, respectively. Any MVK polypeptidethat is feedback-resistant can be used in the compositions and methodsdisclosed herein for the production of isoprene. Exemplary archaeal MVKpolypeptides that are predicted to be feedback-resistant based on theirhomology to the M. mazei MVK polypeptide include YP_(—)304960.1mevalonate kinase Methanosarcina barkeri str. Fusaro, NP_(—)615566.1mevalonate kinase Methanosarcina acetivorans C2A, YP_(—)566996.1mevalonate kinase Methanococcoides burtonii DSM 6242, and YP_(—)684687.1mevalonate kinase uncultured methanogenic archaeon RC-I. For thisprediction, the default parameters were used for a protein-protein BLASTsearch using the NCBI BLAST software currently publicly available on theworld wide web. Additionally, FIG. 138 shows a M. mazei mevalonatekinase BLAST search distance tree that was created using the distancetree of results link on the NCBI BLAST results page. Exemplary potentialfeedback-resistant mevalonate kinase polypeptides that lack the loopdisclosed by Fue et al. are circled. In some embodiments, any of the MVKnucleic acids and polypeptides listed in Appendix 1 in the section“Exemplary mevalonate kinase nucleic acids and polypeptides” that arefeedback-resistant (e.g., MVK polypeptides that lack the loop denoted inFIG. 137) are used in the compositions or methods disclosed herein. Insome embodiments, any of the mutated MVK polypeptides that arefeedback-resistant and that are disclosed by WO 2004/111214 are used.

As used herein, “non-modified nucleic acids encoding feedback-resistantmevalonate kinase” refers to any mevalonate kinase which has not beenmanipulated to increase mevalonate kinase activity to a greater extentthan if no manipulation has occurred. For example, mutation(s) to one ormore nucleotide(s) or amino acids of a mevalonate kinase found in natureis considered to be a modified mevalonate kinase. Examples of modifiedmevalonate kinases are disclosed in WO 2004/111214 and WO 2006/063752.In some embodiments, the modified mevalonate kinases in WO 2004/111214and WO 2006/063752 are excluded from the invention. In otherembodiments, the feedback resistant mevalonate kinases of this inventiondo not include a modified mevalonate kinase wherein at least onemutation is at one or more amino acid position(s) selected from thegroup consisting of amino acid positions corresponding to positions 55,59, 66, 83, 106, 111, 117, 142, 152, 158, 218, 231, 249, 367 and 375 ofthe amino acid sequence of Saccharomyces cerevisiae mevalonate kinase asshown in SEQ ID NO:1 in WO 2006/063752. In another embodiment, thefeedback resistant mevalonate kinases of this invention do not include amodified mevalonate kinase having the nucleotide sequence SEQ ID NO: 5in WO 2006/063752.

Thus, the MVK polypeptide from M. mazei or other feedback-resistant MVKpolypeptides can be used to decrease feedback inhibition by downstreammetabolites of the isoprene or isoprenoid pathways (such as DMAPP, GPP,and/or FPP) and increase the rate of production of DMAPP compared to MVKpolypeptides that are more sensitive to feedback inhibition. If desired,a heterologous nucleic acid or a duplicate copy of an endogenous nucleicacid encoding an isoprene synthase polypeptide can be used to increasethe conversion of DMAPP to isoprene.

In particular, both a high flux from central metabolism to DMAPP and arobust enzyme activity to catalyze the conversion of DMAPP to isopreneare desirable for the commercial scale production of isoprene in vivo.Since high concentrations of DMAPP are growth inhibitory, high fluxthrough the MVA pathway is desirably accompanied by high isoprenesynthase polypeptide activity to avoid accumulation of toxic amounts ofDMAPP. Accordingly, in one aspect, the invention features a method ofproducing isoprene that involves increasing the expression and/oractivity of (i) a MVK polypeptide (such as a feedback-resistant MVKpolypeptide) and (ii) an isoprene synthase polypeptide compared to theexpression level and/or activity level normally found in the cell. Forexample, overexpressing the MVK polypeptide from M. mazei and theisoprene synthase from kudzu supports high flux to DMAPP andsimultaneous conversion of DMAPP to isoprene. Furthermore, by balancingthe activity of the MVK polypeptide and the isoprene synthasepolypeptide, we have generated cells which convert acetyl-CoA toisoprene at high flux and titer without the accumulation of DMAPP. Thetotal activity level of an MVK polypeptide is influenced by both thelevel of protein expressed and the enzymatic characteristics of thespecific MVK polypeptide used. Limiting the accumulation of DMAPP isvaluable because it prevents DMAPP-associated growth inhibition and lossof metabolic activity.

As described further in the Examples, overexpression of thefeedback-resistant M. mazei MVK polypeptide and the kudzu isoprenesynthase polypeptide resulted in an eight-fold increase in isoprenetiter compared to overexpression of isoprene synthase alone. Asdiscussed in Examples 3-5, E. coli cells containing the MVA pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from kudzu (pTrcKudzuMVK(M. mazei)) were used toproduce isoprene in 15-L bioreactors. Example 3 indicates that the totalamount of isoprene produced during a 68 hour fermentation was 227.2 g.Instantaneous volumetric productivity levels reached values as high as1.5 g isoprene/L broth/hr, and the instantaneous yield levels reached ashigh as 17.7% w/w (Example 4). Example 5 indicates that the molar yieldof utilized carbon that went into producing isoprene during thisfermentation was 16.6%, and the weight percent yield of isoprene fromglucose over the entire fermentation was 7.7%. Example 9 indicates thatoverexpression of the feedback-inhibited S. cerevisiae MVK polypeptideproduced less isoprene than overexpression of the feedback-resistant M.mazei MVK polypeptide.

Example 6 describes the comparison of four strains with differentrelative levels of isoprene synthase polypeptide activity and MVKpolypeptide activity: (i) the MCM343 strain with low MVK polypeptideactivity and high isoprene synthase polypeptide activity, (ii) theMCM401 strain with high MVK polypeptide activity and high isoprenesynthase polypeptide activity, (iii) the MCM437 with low MVK polypeptideactivity and low isoprene synthase, and (iv) the MCM438 strain with highMVK polypeptide activity and low isoprene synthase polypeptide activity.In particular, the specific productivity of isoprene from a strainexpressing the full mevalonic acid pathway and kudzu isoprene synthasepolypeptide at low levels (MCM437) was compared to a strain that inaddition over-expressed MVK polypeptide from M. mazei and kudzu isoprenesynthase polypeptide (MCM401), as well as strains that eitherover-expressed just MVK polypeptide (MCM438), or just kudzu isoprenesynthase polypeptide (MCM343). The strain over-expressing both MVKpolypeptide and isoprene synthase polypeptide (MCM401) had higherspecific productivity of isoprene compared to the strain over-expressingjust MVK polypeptide (MCM438) or just kudzu isoprene synthasepolypeptide (MCM343). The strain with low activities of both MVKpolypeptide and kudzu isoprene synthase polypeptide (MCM437) had thelowest specific productivity of isoprene overall.

Accordingly, in some embodiments, the cells overexpress both an MVKpolypeptide (such as a feedback-resistant MVK polypeptide) and anisoprene synthase polypeptide. In the experiments described in Examples2-5, E. coli cells containing the upper mevalonic acid (MVA) pathway(pCL PtrcUpperPathway encoding E. faecalis mvaE and mvaS), theintegrated lower MVA pathway (gi1.2KKDyI encoding S. cerevisiaemevalonate kinase, mevalonate phosphate kinase, mevalonate pyrophosphatedecarboxylase, and IPP isomerase), and high expression of mevalonatekinase from M. mazei and isoprene synthase from kudzu (pTrcKudzuMVK(M.mazei)0 were used to produce isoprene. In these experiments, the M.mazei MVK polypeptide and kudzu isoprene synthase polypeptide wereoverexpressed from a high copy plasmid under the control of a strongpromoter. In contrast, the S. cerevisiae lower MVA pathway nucleic acids(mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase) were present as a singlecopy of the nucleic acids integrated in the chromosome under the controlof a weak promoter. The E. faecalis upper MVA pathway nucleic acids(mvaE encoding a naturally occurring fusion protein that has bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoAreductase activities and mvaS encoding a 3-hydroxy-3-methylglutaryl-CoAsynthase polypeptide) were overexpressed from a medium copy plasmidunder the control of a strong promoter (the same promoter used toexpress the M. mazei MVK polypeptide and kudzu isoprene synthasepolypeptide). Thus, the M. mazei MVK polypeptide and kudzu isoprenesynthase polypeptide were expressed at a much higher level than theother MVA pathway polypeptides. Since the feedback-resistant M. mazeiMVK polypeptide was expressed at a much higher level than thefeedback-inhibited S. cerevisiae MVK polypeptide, most of the conversionof MVA to MVAP seems to be due to the M. mazei MVK polypeptide ratherthan the S. cerevisiae MVK polypeptide. If desired, the S. cerevisiaeMVK nucleic acid can be removed from any of the cells disclosed hereinusing standard methods (such that the only heterologous MVK nucleic acidis the M. mazei MVK nucleic acid). If desired, the S. cerevisiae MVKnucleic acid can alternatively be replaced by any other MVK nucleic acidin any of the cells described herein.

Accordingly, in some embodiments, an MVK polypeptide (such as afeedback-resistant MVK polypeptide) and/or an isoprene synthasepolypeptide is expressed at a level that is at least about any of 2, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275,300, 350, 400, 450, or 500-fold (i) higher than the level of expressionof a second MVA pathway polypeptide (such as an acetyl-CoAacetyltransferase (AACT) polypeptide, 3-hydroxy-3-methylglutaryl-CoAsynthase (HMGS) polypeptide, 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR) polypeptide, phosphomevalonate kinase (PMK) polypeptide,diphosphomevalonate decarboxylase (DPMDC) polypeptide, orisopentenyl-diphosphate delta-isomerase (IDI) polypeptide) or (ii)higher than the level of expression of all other MVA pathwaypolypeptides in the cell. In particular embodiments, the MVK polypeptideand/or an isoprene synthase polypeptide is expressed a level that is atleast about any of 2, 5, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80,90, 100, 125, 150, 200, 225, 250, 275, 300, 350, 400, 450, or 500-foldhigher than the level of expression of an AACT polypeptide, HMGSpolypeptide, and HMGR polypeptide. In particular embodiments, the MVKpolypeptide and/or an isoprene synthase polypeptide is expressed a levelthat is at least about any of 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 200, 225, 250, 275, 300, 350, 400, 450, or 500-foldhigher than the level of expression of an PMK polypeptide, DPMDCpolypeptide, and IDI polypeptide. In some embodiments, the total amountof MVK polypeptide is similar to the total amount of isoprene synthasepolypeptide. For example, in some embodiments, the total amount of MVKpolypeptide is within about any of 10, 8, 6, 4, 2, 1, or 0.5-fold higheror lower than the total amount of isoprene synthase polypeptide (e.g.,the amount of MVK polypeptide may be between about 10-fold lower toabout 10-fold higher than the amount of isoprene synthase polypeptide).Standard methods (such as western blotting) can be used to quantitatethe amount of any of these polypeptides. Standard methods can be used toalter the relative amounts of expressed MVA pathway polypeptides, suchas by using a stronger promoter or a plasmid with a higher copy numberto express an MVK polypeptide and/or an isoprene synthase polypeptidecompared to the promoter(s) and plasmid(s) used to express other MVApathway polypeptides.

In some embodiments, an MVK RNA molecule (such as an RNA moleculeencoding a feedback-resistant polypeptide) and/or an isoprene synthaseRNA molecule is expressed at a level that is at least about any of 2, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275,300, 350, 400, 450, or 500-fold (i) higher than the level of expressionof a second MVA pathway RNA molecule (such as an AACT RNA molecule, HMGSRNA molecule, HMGR RNA molecule, PMK RNA molecule, DPMDC RNA molecule,or IDI RNA molecule) or (ii) higher than the level of expression of allother MVA pathway RNA molecules in the cell. In particular embodiments,the MVK RNA molecule and/or an isoprene synthase RNA molecule isexpressed at a level that is at least about any of 2, 5, 10, 12, 14, 16,18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275,300, 350, 400, 450, or 500-fold higher than the level of expression ofan AACT RNA molecule, HMGS RNA molecule, and HMGR RNA molecule. Inparticular embodiments, the MVK RNA molecule and/or an isoprene synthaseRNA molecule is expressed at a level that is at least about any of 2, 5,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275,300, 350, 400, 450, or 500-fold higher than the level of expression ofan PMK RNA molecule, DPMDC RNA molecule, and IDI RNA molecule. In someembodiments, the total amount of MVK RNA is similar to the total amountof isoprene synthase RNA. For example, in some embodiments, the totalamount of MVK RNA is within about any of 10, 8, 6, 4, 2, 1, or 0.5-foldhigher or lower than the total amount of isoprene synthase RNA (e.g.,the amount of MVK RNA may be between about 10-fold lower to about10-fold higher than the amount of isoprene synthase RNA). Standardmethods (such as northern blotting) can be used to quantitate the amountof any of these RNA molecules. Standard methods can be used to alter therelative amounts of expressed MVA pathway RNA molecules, such as byusing a stronger promoter or a plasmid with a higher copy number toexpress an MVK RNA molecule and/or an isoprene synthase RNA moleculecompared to the promoter(s) and plasmid(s) used to express other MVApathway RNA molecules.

In some embodiments, the number of copies of an MVK DNA molecule (suchas a DNA molecule encoding a feedback-resistant polypeptide) and/or anisoprene synthase DNA molecule is at least about any of 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275, 300, 350,400, 450, or 500-fold (i) higher than the number of copies of a secondMVA pathway DNA molecule (such as an AACT DNA molecule, HMGS DNAmolecule, HMGR DNA molecule, PMK DNA molecule, DPMDC DNA molecule, orIDI DNA molecule) or (ii) higher than the number of copies of all otherMVA pathway DNA molecules in the cell. In particular embodiments, thenumber of copies of an MVK DNA molecule and/or an isoprene synthase DNAmolecule is at least about any of 2, 5, 10, 12, 14, 16, 18, 20, 30, 40,50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275, 300, 350, 400,450, or 500-fold higher than the number of copies of an AACT DNAmolecule, HMGS DNA molecule, and HMGR DNA molecule. In particularembodiments, the number of copies of a MVK DNA molecule and/or anisoprene synthase DNA molecule is at least about any of 2, 5, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 225, 250, 275, 300, 350,400, 450, or 500-fold higher than the number of copies of an PMK DNAmolecule, DPMDC DNA molecule, and IDI DNA molecule. In some embodiments,the number of copies of an MVK DNA molecule is similar to the number ofcopies of an isoprene synthase DNA molecule. For example, in someembodiments, the number of copies of an MVK DNA molecule is within aboutany of 10, 8, 6, 4, 2, 1, or 0.5-fold higher or lower than the number ofcopies of an isoprene synthase DNA molecule (e.g., the number of copiesof a MVK DNA may be between about 10-fold lower to about 10-fold higherthan the number of copies of an isoprene synthase DNA molecule).Standard methods (such as southern blotting) can be used to quantitatethe amount of any of these DNA molecules. Standard methods can be usedto alter the relative amounts of MVA pathway DNA molecules, such as byusing a plasmid with a higher copy number to insert an MVK DNA moleculeand/or an isoprene synthase DNA molecule compared to the plasmid(s) usedto insert other MVA pathway DNA molecules.

As discussed above, using a feedback-resistant MVK polypeptide decreasesthat amount of MVA that accumulates in the cell medium since more MVA isconverted to MVAP. Increasing the expression of an isoprene synthasepolypeptide decreases the accumulation of DMAPP since more DMAPP isconverted to isoprene. If desired, the expression of a PMK polypeptide,DPMDC polypeptide, IDI polypeptide, or any combination of two or more ofthe foregoing can also be increased to reduce the accumulation of MVApathway intermediates and/or to increase the flux through the MVApathway. In some embodiments, the amount of MVA, DMAPP, IPP, GPP, FPP,or any combination of two or more of the foregoing allows production ofisoprene without causing undesirable amounts of growth inhibition,toxicity, or cell death. In some embodiments, the amount of MVA, DMAPP,and/or IPP is high enough to allow production of isoprene in any of theamounts or concentrations disclosed below in the “Exemplary Productionof Isoprene” section. In some embodiments, a detectable amount of MVA,DMAPP, and/or IPP does not accumulate since the intermediate(s) arebeing converted to downstream molecules at a rate that does not allow adetectable amount of MVA, DMAPP, and/or IPP to accumulate. Example 8,parts IV and V indicate that overexpression of the feedback-inhibited S.cerevisiae MVK polypeptide is correlated with the accumulation of moreDMAPP and IPP than overexpression of the feedback-resistant M. mazei MVKpolypeptide. This accumulation of DMAPP can cause undesirable growthinhibition. A goal is therefore to achieve a pathway enzyme balance tominimize the accumulation of these metabolites for the relief of growthinhibition.

Tables 15A and 15B list exemplary desirable concentrations of DMAPP,IPP, GPP, and FPP as well as examples of relatively high concentrationsof these metabolites that have been detected using the cells and methodsdescribed herein. Table 15B has the same data as Table 15A that has beennormalized to grams of dry cell weight assuming that 1 liter of theculture at OD=1 has 0.33 grams dry cell weight (g_(dcw)). For theseexperiments, the quantitation limit is below 0.1 mM for theintracellular concentrations of DMAPP, FPP, GPP, and IPP. If desired,more sensitive equipment can be used to detect even smaller amounts ofthese compounds. The lowest absolute concentrations that were used asstandards for the LCMS analysis were 3.4 uM DMAPP, 1.7 uM IPP, 0.9 uMGPP, and 2.3 uM FPP. Thus, absolute amounts that are equal to or greaterthan these standard amounts can be readily quantified.

In these experiments, there was a negligible amount of DMAPP, FPP, GPP,and IPP in the liquid cell medium (outside of the cells). Thus, theamounts listed in Tables 15A and 15B are representative of theintracellular concentrations of DMAPP, FPP, GPP, and IPP.

TABLE 15A Exemplary metabolite concentrations Metabolite DMAPP IPP GPPFPP Intracellular Exemplary 0.4 mM¹ 0.3 mM¹ 0.7 mM² 1.4 mM¹concentration, desirable mM concentrations Exemplary 9.2 mM³ 27-40 mM⁴ 2.8 mM³ 3.6 mM³ detected 15.3 mM⁵  6.3 mM⁵ 3.3 mM⁵ concentrations¹Example 3. ²Example 8, Part VII. ³Example 7, Part III. ⁴Example 8, PartVIII. ⁵Example 7, Part II.

TABLE 15B Exemplary metabolite concentrations Metabolite DMAPP IPP GPPFPP Intracellular Exemplary 0.3¹ 0.2¹ 0.5² 1.1¹ concentration, desirableμmol/g_(dcw) ⁶ concentrations Exemplary 7.0³ 20-30⁴ 2.1³ 2.0³ detected11.6⁵ 4.8⁵ 3.3⁵ concentrations ¹Example 3. ²Example 8, Part VII.³Example 7, Part III. ⁴Example 8, Part VIII. ⁵Example 7, Part II.

In some embodiments, the intracellular concentration of DMAPP is betweenabout 0 to about 25 μmol/g_(dcw), such as between about 0.1 to about 20μmol g_(dcw), about 0.1 to about 15 μmol/g_(dcw), about 0.1 to about 11μmol/g_(dcw), about 0.1 to about 7 μmol/g_(dcw), about 0.1 to about 5μmol/g_(dcw), about 0.1 to about 2 μmol/g_(dcw), about 0.1 to about 1μmol/g_(dcw), about 0.1 to about 0.8 μmol/g_(dcw), about 0.1 to about0.6 μmol/g_(dcw), about 0.2 to about 15 μmol/g_(dcw), about 0.2 to about11 μmol/g_(dcw), about 0.2 to about 7 μmol/g_(dcw), about 0.2 to about 5μmol/g_(dcw), about 0.2 to about 2 μmol/g_(dcw), about 0.3 to about 11μmol/g_(dcw), about 0.3 to about 7 μmol/g_(dcw), about 0.3 to about 5μmol/g_(dcw), about 0.3 to about 2 μmol/g_(dcw), about 0.3 to about 1μmol/g_(dcw), about 0.4 to about 11 μmol/g_(dcw), about 0.4 to about 7μmol/g_(dcw), about 0.4 to about 5 μmol/g_(dcw), about 0.4 to about 2μmol/g_(dcw), about 0.5 to about 7 μmol/g_(dcw), about 0.5 to about 5μmol/g_(dcw), or about 0.5 to about 2 μmol/g_(dcw). In some embodiments,the intracellular concentration of DMAPP is equal to or less than aboutany of 25, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.8, 0.6, 0.5, 0.4,0.3, 0.2, or 0.1 μmol/g_(dcw).

In some embodiments, the intracellular concentration of IPP is betweenabout 0 to about 60 μmol/g_(dcw), such as between about 0.1 to about 50μmol/g_(dcw), about 0.1 to about 40 μmol/g_(dcw), about 0.1 to about 30μmol/g_(dcw), about 0.1 to about 20 μmol/g_(dcw), about 0.1 to about 15μmol/g_(dcw), about 0.1 to about 11 μmol/g_(dcw), about 0.1 to about 7μmol/g_(dcw), about 0.1 to about 5 μmol/g_(dcw), about 0.1 to about 2μmol/g_(dcw), about 0.1 to about 1 μmol/g_(dcw), about 0.1 to about 0.8μmol/g_(dcw), about 0.1 to about 0.6 μmol/g_(dcw), about 0.2 to about 60μmol/g_(dcw), about 0.2 to about 50 μmol/g_(dcw), about 0.2 to about 40μmol/g_(dcw), about 0.2 to about 30 μmol/g_(dcw), about 0.2 to about 20μmol/g_(dcw), about 0.2 to about 15 μmol/g_(dcw), about 0.2 to about 11μmol/g_(dcw), about 0.2 to about 7 μmol/g_(dcw), about 0.2 to about 5μmol/g_(dcw), about 0.2 to about 2 μmol/g_(dcw), about 0.3 to about 60μmol/g_(dcw), about 0.3 to about 50 μmol/g_(dcw), about 0.3 to about 40μmol/g_(dcw), about 0.3 to about 30 μmol/g_(dcw), about 0.3 to about 15μmol/g_(dcw), about 0.3 to about 11 μmol/g_(dcw), about 0.3 to about 7μmol/g_(dcw), about 0.3 to about 5 μmol/g_(dcw), about 0.3 to about 2μmol/g_(dcw), about 0.4 to about 60 μmol/g_(dcw), about 0.4 to about 50μmol/g_(dcw), about 0.4 to about 40 μmol/g_(dcw), about 0.4 to about 30μmol/g_(dcw), about 0.4 to about 15 μmol/g_(dcw), about 0.4 to about 7μmol/g_(dcw), about 0.4 to about 5 μmol/g_(dcw), about 0.4 to about 2μmol/g_(dcw), about 0.5 to about 60 μmol/g_(dcw), about 0.5 to about 50μmol/g_(dcw), about 0.5 to about 40 μmol/g_(dcw), about 0.5 to about 30μmol/g_(dcw), about 0.5 to about 15 μmol/g_(dcw), about 0.5 to about 11μmol/g_(dcw), about 0.5 to about 7 μmol/g_(dcw), about 0.5 to about 5μmol/g_(dcw), or about 0.5 to about 2 μmol/g_(dcw). In some embodiments,the intracellular concentration of IPP is equal to or less than aboutany of 60, 50, 40, 30, 25, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.8,0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μmol/g_(dcw).

In some embodiments, the intracellular concentration of GPP is betweenabout 0 to about 8 μmol/g_(dcw), such as between about 0.1 to about 7μmol/g_(dcw), about 0.1 to about 6 μmol/g_(dcw), about 0.1 to about 5μmol/g_(dcw), about 0.1 to about 4 μmol/g_(dcw), about 0.1 to about 3μmol/_(dcw), about 0.1 to about 2 μmol/g_(dcw), about 0.1 to about 1μmol/g_(dcw), about 0.1 to about 0.8 μmol/g_(dcw), about 0.1 to about0.6 μmol/g_(dcw), about 0.2 to about 7 μmol/g_(dcw), about 0.2 to about6 μmol/g_(dcw), about 0.2 to about 5 μmol/g_(dcw), about 0.2 to about 4μmol/g_(dcw), about 0.2 to about 3 μmol/g_(dcw), about 0.2 to about 2μmol/g_(dcw), about 0.3 to about 7 μmol/g_(dcw), about 0.3 to about 6μmol/g_(dcw), about 0.3 to about 5 μmol/g_(dcw), about 0.3 to about 4μmol/g_(dcw), about 0.3 to about 3 μmol/g_(dcw), about 0.3 to about 2μmol/g_(dcw), about 0.4 to about 7 μmol/g_(dcw), about 0.4 to about 6μmol/g_(dcw), about 0.4 to about 5 μmol/g_(dcw), about 0.4 to about 2μmol/g_(dcw), about 0.5 to about 7 μmol/g_(dcw), about 0.5 to about 5μmol/g_(dcw), about 0.5 to about 2 μmol/g_(dcw), about 0.6 to about 7μmol/g_(dcw), about 0.6 to about 5 μmol/g_(dcw), about 0.6 to about 2μmol/g_(dcw), about 0.7 to about 7 μmol/g_(dcw), about 0.7 to about 5μmol/g_(dcw), or about 0.7 to about 2 μmol/g_(dcw). In some embodiments,the intracellular concentration of GPP is equal to or less than aboutany of 8, 6, 4, 2, 1, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 μmol/g_(dcw).

In some embodiments, the intracellular concentration of FPP is betweenabout 0 to about 6 μmol/g_(dcw), such as between about 0.1 to about 6μmol/g_(dcw), about 0.1 to about 5 μmol/g_(dcw), about 0.1 to about 4μmol/g_(dcw), about 0.1 to about 3 μmol/g_(dcw), about 0.1 to about 2μmol/g_(dcw), about 0.1 to about 1 μmol/g_(dcw), about 0.1 to about 0.8μmol/g_(dcw), about 0.1 to about 0.6 μmol/g_(dcw), about 0.2 to about 6μmol/g_(dcw), about 0.2 to about 5 μmol/g_(dcw), about 0.2 to about 4μmol/g_(dcw), about 0.2 to about 3 μmol/g_(dcw), about 0.2 to about 2μmol/g_(dcw), about 0.3 to about 6 μmol/g_(dcw), about 0.3 to about 5μmol/g_(dcw), about 0.3 to about 4 μmol/g_(dcw), about 0.3 to about 3μmol/g_(dcw), about 0.3 to about 2 μmol/g_(dcw), about 0.4 to about 6μmol/g_(dcw), about 0.4 to about 5 μmol/g_(dcw), about 0.4 to about 2μmol/g_(dcw), about 0.5 to about 6 μmol/g_(dcw), about 0.5 to about 5μmol/g_(dcw), about 0.5 to about 2 μmol/g_(dcw), about 0.8 to about 6μmol/g_(dcw), about 0.8 to about 5 μmol/g_(dcw), about 0.8 to about 2μmol/g_(dcw), about 1 to about 6 μmol/g_(dcw), about 1 to about 5μmol/g_(dcw), about 1 to about 2 μmol/g_(dcw), about 1.1 to about 6μmol/g_(dcw), about 1.1 to about 5 μmol/g_(dcw), about 1.1 to about 2μmol/g_(dcw), about 1.1 to about 1.5 μmol/g_(dcw), about 1.2 to about 6μmol/g_(dcw), about 1.2 to about 5 μmol/g_(dcw), about 1.2 to about 2μmol/g_(dcw), or about 1.2 to about 1.5 μmol/g_(dcw). In someembodiments, the intracellular concentration of FPP is equal to or lessthan about any of 6, 4, 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.6,0.5, 0.4, 0.3, 0.2, or 0.1 μmol/g_(dcw).

In some embodiments, the concentration (e.g., concentration in the cellmedium) of MVA is between about 0 to about 120 g/L, such as betweenabout about 0 to about 110 g/L, such as between about 0.1 to about 100g/L, about 0.1 to about 75 g/L, about 0.1 to about 60 g/L, about 0.1 toabout 50 g/L, about 0.1 to about 40 g/L, about 0.1 to about 30 g/L,about 0.1 to about 20 g/L, about 0.1 to about 15 g/L, about 0.1 to about11 g/L, about 0.1 to about 7 g/L, about 0.1 to about 5 g/L, about 0.1 toabout 2 g/L, about 0.1 to about 1 g/L, about 0.1 to about 0.8 g/L, about0.1 to about 0.6 g/L, about 0.2 to about 120 g/L, about 0.2 to about 100g/L, about 0.2 to about 75 g/L, about 0.2 to about 60 g/L, about 0.2 toabout 50 g/L, about 0.2 to about 40 g/L, about 0.2 to about 30 g/L,about 0.2 to about 20 g/L, about 0.2 to about 15 g/L, about 0.2 to about11 g/L, about 0.2 to about 7 g/L, about 0.2 to about 5 g/L, about 0.2 toabout 2 g/L, about 0.3 to about 120 g/L, about 0.3 to about 100 g/L,about 0.3 to about 75 g/L, about 0.3 to about 60 g/L, about 0.3 to about50 g/L, about 0.3 to about 40 g/L, about 0.3 to about 30 g/L, about 0.3to about 15 g/L, about 0.3 to about 11 g/L, about 0.3 to about 7 g/L,about 0.3 to about 5 g/L, about 0.3 to about 2 g/L, about 0.4 to about120 g/L, about 0.4 to about 100 g/L, about 0.4 to about 75 g/L, about0.4 to about 60 g/L, about 0.4 to about 50 g/L, about 0.4 to about 40g/L, about 0.4 to about 30 g/L, about 0.4 to about 15 g/L, about 0.4 toabout 7 g/L, about 0.4 to about 5 g/L, about 0.4 to about 2 g/L, about0.5 to about 1200 g/L, about 0.5 to about 100 g/L, about 0.5 to about 75g/L, about 0.5 to about 60 g/L, about 0.5 to about 50 g/L, about 0.5 toabout 40 g/L, about 0.5 to about 30 g/L, about 0.5 to about 15 g/L,about 0.5 to about 11 g/L, about 0.5 to about 7 g/L, about 0.5 to about5 g/L, about 0.5 to about 2 g/L, about 50 to about 60 g/L, or about 1g/L. In some embodiments, the concentration (e.g., concentration in thecell medium) of MVA is equal to or less than about any of 120, 100, 80,70, 60, 50, 40, 30, 25, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.8, 0.6,0.5, 0.4, 0.3, 0.2, or 0.1 g/L.

Examples 10-21 also support the use of the compositions and methodsdisclosed herein to produce large amounts of isoprene. The methodsdescribed herein can be used to modify any of the cells and methods ofExamples 10-21 to express a feedback-resistant MVK polypeptide (such asa M. mazei MVK polypeptide) either as the only MVK polypeptide or as anadditional MVK polypeptide. Additionally, methods described herein canbe used to modify any of the cells and methods of U.S. Ser. No.61/134,094, filed Jul. 2, 2008 (which is hereby incorporated byreference in its entirety, particularly with respect to methods ofmaking isoprene and isoprene compositions) to express afeedback-resistant MVK polypeptide (such as a M. mazei MVK polypeptide)either as the only MVK polypeptide or as an additional MVK polypeptide.As discussed above, the use of a feedback-resistant MVK polypeptide mayfurther increase the production of isoprene.

Summary of Exemplary Compositions and Methods for Producing Isoprene

This section summaries exemplary compositions and methods for producingisoprene that can be used with a feedback-resistant MVK polypeptide(such as a M. mazei MVK polypeptide). In one aspect, the inventionfeatures compositions and methods for the production of isoprene inincreased amounts and/or purity. In one aspect, compositions and methodsof the invention increase the rate of isoprene production and increasethe total amount of isoprene that is produced. For example, cell culturesystems that generate 4.8×10⁴ nmole/g_(wcm)/hr of isoprene have beenproduced (Table 1). The efficiency of these systems is demonstrated bythe conversion of about 2.2% of the carbon that the cells consume from acell culture medium into isoprene. As shown in the Examples and Table 2,approximately 3 g of isoprene per liter of broth was generated. Ifdesired, even greater amounts of isoprene can be obtained using otherconditions, such as those described herein. In some embodiments, arenewable carbon source is used for the production of isoprene. In someembodiments, the production of isoprene is decoupled from the growth ofthe cells. In some embodiments, the concentrations of isoprene and anyoxidants are within the nonflammable ranges to reduce or eliminate therisk that a fire may occur during production or recovery of isoprene.The compositions and methods of the present invention are desirablebecause they allow high isoprene yield per cell, high carbon yield, highisoprene purity, high productivity, low energy usage, low productioncost and investment, and minimal side reactions. This efficient, largescale, biosynthetic process for isoprene production provides an isoprenesource for synthetic isoprene-based rubber and provides a desirable,low-cost alternative to using natural rubber.

As discussed further herein, the amount of isoprene produced by cellscan be greatly increased by introducing a heterologous nucleic acidencoding an isoprene synthase polypeptide (e.g., a plant isoprenesynthase polypeptide) into the cells. Isoprene synthase polypeptidesconvert dimethylallyl diphosphate (DMAPP) into isoprene. As shown in theExamples, a heterologous Pueraria Montana (kudzu) isoprene synthasepolypeptide was expressed in a variety of host cells, such asEscherichia coli, Panteoa citrea, Bacillus subtilis, Yarrowialipolytica, and Trichoderma reesei. All of these cells produced moreisoprene than the corresponding cells without the heterologous isoprenesynthase polypeptide. As illustrated in Tables 1 and 2, large amounts ofisoprene are produced using the methods described herein. For example,B. subtilis cells with a heterologous isoprene synthase nucleic acidproduced approximately 10-fold more isoprene in a 14 liter fermentorthan the corresponding control B. subtilis cells without theheterologous nucleic acid (Table 2). The production of 300 mg ofisoprene per liter of broth (mg/L, wherein the volume of broth includesboth the volume of the cell medium and the volume of the cells) by E.coli and 30 mg/L by B. subtilis in fermentors indicates that significantamounts of isoprene can be generated (Table 2). If desired, isoprene canbe produced on an even larger scale or other conditions described hereincan be used to further increase the amount of isoprene. The vectorslisted in Tables 1 and 2 and the experimental conditions are describedin further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example 10, part II. For this assay, a samplewas removed at one or more time points from the shake flask and culturedfor 30 minutes. The amount of isoprene produced in this sample was thenmeasured. The headspace concentration and specific rare of isopreneproduction are listed in Table 1 and described further herein. IsopreneProduction in a Headspace vial* Headspace Specific Rate concentrationμg/L_(broth)/hr/OD Strain μg/L_(gas) (nmol/g_(wcm)/hr) E. coliBL21/pTrcKudzu IS 1.40 53.2 (781.2) E. coli BL21/pCL DXS yidi Kudzu 7.61289.1 (4.25 × 10³) IS E. coli BL21/MCM127 with kudzu 23.0 874.1 (12.8 ×10³) IS and entire MVA pathway E. coli BL21/pET N-HisKudzu IS 1.49 56.6(831.1) Pantoea citrea/pTrcKudzu IS 0.66 25.1 (368.6) E. coli w/PoplarIS [Miller (2001)] — 5.6 (82.2) Bacillis licheniformis Fall — 4.2 (61.4)U.S. Pat. No. 5,849,970 Yarrowia lipolytica with kudzu ~0.05 μg/L ~2(~30) isoprene synthase Trichoderma reesei with kudzu ~0.05 μg/L ~2(~30) isoprene synthase E. coli BL21/pTrcKKD_(y)I_(k)IS with 85.9 3.2 ×10³ (4.8 × 10⁴) kudzu IS and lower MVA pathway *Normalized to 1 mL of 1OD₆₀₀, cultured for 1 hour in a sealed headspace vial with a liquid toheadspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cellcultures and methods of the invention. The assay for measuring isopreneproduction is described in Example 10, part II. For this assay, a sampleof the off-gas of the fermentor was taken and analyzed for the amount ofisoprene. The peak headspace concentration (which is the highestheadspace concentration during the fermentation), titer (which is thecumulative, total amount of isoprene produced per liter of broth), andpeak specific rate of isoprene production (which is the highest specificrate during the fermentation) are listed in Table 2 and describedfurther herein. Isoprene Production in Fermentors Peak Peak SpecificHeadspace rate concentration** Titer μg/L_(broth)/hr/OD Strain(ug/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr) E. coli BL21/pTrcKudzu 5241.2 37 with Kudzu IS (543.3) E. coli FM5/pTrcKudzu 3 3.5 21.4 IS(308.1) E. coli BL21/triple strain 285 300 240 (DXS, yidi, IS) (3.52 ×10³) E. coli FM5/triple strain 50.8 29 180.8 (DXS, yidi, IS) (2.65 ×10³) E. coli/MCM127 with 3815 3044 992.5 Kudzu IS and entire (1.46 ×10⁴) MVA pathway E. coli BL21/pCLPtrc 2418 1640 1248 UpperPathway gi1.2(1.83 × 10⁴) integrated lower pathway pTrcKudzu E. coli BL21/MCM40113991 23805 3733 with 4 × 50 uM IPTG (5.49 × 10⁴) E. coli BL21/MCM40122375 19541 5839.5 with 2 × 100 uM IPTG (8.59 × 10⁴) E. coliBL21/pCLPtrc 3500 3300 1088 UpperPathwayHGS2 - (1.60 × 10⁴) pTrcKKDyIkISBacillus subtilis 1.5 2.5 0.8 wild-type (11.7) Bacillus pBS Kudzu IS16.6 ~30 5 (over 100 hrs) (73.4) Bacillus Marburg 6051 2.04 0.61 24.5[Wagner and Fall (1999)] (359.8) Bacillus Marburg 6051 0.7 0.15 6.8 FallU.S. Pat. (100) No. 5,849,970 **Normalized to an off-gas flow rate of 1vvm (1 volume off-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologousisoprene synthase nucleic acid can be enhanced by increasing the amountof a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or anisopentenyl diphosphate isomerase (IDI) polypeptide expressed by thecells. For example, a DXS nucleic acid and/or an IDI nucleic acid can beintroduced into the cells. The DXS nucleic acid may be a heterologousnucleic acid or a duplicate copy of an endogenous nucleic acid.Similarly, the IDI nucleic acid may be a heterologous nucleic acid or aduplicate copy of an endogenous nucleic acid. In some embodiments, theamount of DXS and/or IDI polypeptide is increased by replacing theendogenous DXS and/or IDI promoters or regulatory regions with otherpromoters and/or regulatory regions that result in greater transcriptionof the DXS and/or IDI nucleic acids. In some embodiments, the cellscontain both a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for thebiosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvateand D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate.While not intending to be bound by any particular theory, it is believedthat increasing the amount of DXS polypeptide increases the flow ofcarbon through the DXP pathway, leading to greater isoprene production.IDI polypeptides catalyze the interconversion of isopentenyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP). While not intending to bebound by any particular theory, it is believed that increasing theamount of IDI polypeptide in cells increases the amount (and conversionrate) of IPP that is converted into DMAPP, which in turn is convertedinto isoprene.

For example, fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used toproduce isoprene. The levels of isoprene varied from 50 to 300 μg/L overa time period of 15 hours (Example 16, part VII).

In some embodiments, the presence of heterologous or extra endogenousisoprene synthase, IDI, and DXS nucleic acids causes cells to grow morereproducibly or remain viable for longer compared to the correspondingcell with only one or two of these heterologous or extra endogenousnucleic acids. For example, cells containing heterologous isoprenesynthase, IDI, and DXS nucleic acids grew better than cells with onlyheterologous isoprene synthase and DXS nucleic acids or with only aheterologous isoprene synthase nucleic acid. Also, heterologous isoprenesynthase, IDI, and DXS nucleic acids were successfully operably linkedto a strong promoter on a high copy plasmid that was maintained by E.coli cells, suggesting that large amounts of these polypeptides could beexpressed in the cells without causing an excessive amount of toxicityto the cells. While not intending to be bound to a particular theory, itis believed that the presence of heterologous or extra endogenousisoprene synthase and IDI nucleic acids may reduce the amount of one ormore potentially toxic intermediates that would otherwise accumulate ifonly a heterologous or extra endogenous DXS nucleic acid was present inthe cells.

In some embodiments, the production of isoprene by cells by cells thatcontain a heterologous isoprene synthase nucleic acid is augmented byincreasing the amount of a MVA pathway polypeptide expressed by thecells (FIGS. 19A and 19B). Exemplary MVA pathways polypeptides includeany of the following polypeptides: acetyl-CoA acetyltransferase (AA-CoAthiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoAsynthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase(HMG-CoA reductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonatedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDIpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of two or more MVA pathway polypeptides. For example, one ormore MVA pathway nucleic acids can be introduced into the cells. In someembodiments, the cells contain the upper MVA pathway, which includesAA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids.In some embodiments, the cells contain the lower MVA pathway, whichincludes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, thecells contain an entire MVA pathway that includes AA-CoA thiolase,HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleicacids. In some embodiments, the cells contain an entire MVA pathway thatincludes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK,PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may beheterologous nucleic acids or duplicate copies of endogenous nucleicacids. In some embodiments, the amount of one or more MVA pathwaypolypeptides is increased by replacing the endogenous promoters orregulatory regions for the MVA pathway nucleic acids with otherpromoters and/or regulatory regions that result in greater transcriptionof the MVA pathway nucleic acids. In some embodiments, the cells containboth a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzuisoprene synthase polypeptide and nucleic acids encoding Saccharomycescerevisia MVK, PMK, MVD, and IDI polypeptides generated isoprene at arate of 6.67×10⁻⁴ mol/L_(broth)/OD₆₀₀/hr (see Example 17). Additionally,a 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid (anintermediate of the MVA pathway). A shake flask of these cells produced2-4 grams of mevalonic acid per liter. These results indicate thatheterologous MVA pathways nucleic acids are active in E. coli. E. colicells that contain nucleic acids for both the upper MVA pathway and thelower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127)produced significantly more isoprene (874 ug/L) compared to E. colicells with nucleic acids for only the lower MVA pathway and the kudzuisoprene synthase (strain MCM 131) (see Table 10 and Example 17, partVIII).

In some embodiments, at least a portion of the cells maintain theheterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the heterologous or duplicate copy of anendogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acidalso comprises a selective marker, such as a kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 16, part VI, the amount of isoprene produced canbe further increased by adding yeast extract to the cell culture medium.In this example, the amount of isoprene produced was linearlyproportional to the amount of yeast extract in the cell medium for theconcentrations tested (FIG. 48C). Additionally, approximately 0.11 gramsof isoprene per liter of broth was produced from a cell medium withyeast extract and glucose (Example 16, part VIII). Both of theseexperiments used E. coli cells with kudzu isoprene synthase, S.cerevisia IDI, and E. coli DXS nucleic acids to produce isoprene.Increasing the amount of yeast extract in the presence of glucoseresulted in more isoprene being produced than increasing the amount ofglucose in the presence of yeast extract. Also, increasing the amount ofyeast extract allowed the cells to produce a high level of isoprene fora longer length of time and improved the health of the cells.

Isoprene production was also demonstrated using three types ofhydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as thecarbon source. E. coli cells with kudzu isoprene synthase, S. cerevisiaIDI, and E. coli DXS nucleic acids produced as much isoprene from thesehydrolyzed biomass carbon sources as from the equivalent amount ofglucose (e.g., 1% glucose, w/v). If desired, any other biomass carbonsource can be used in the compositions and methods of the invention.Biomass carbon sources are desirable because they are cheaper than manyconventional cell mediums, thereby facilitating the economicalproduction of isoprene.

Additionally, invert sugar was shown to function as a carbon source forthe generation of isoprene. For example, 2.4 g/L of isoprene wasproduced from cells expressing MVA pathway polypeptides and a Kudzuisoprene synthase. Glycerol was as also used as a carbon source for thegeneration of 2.2 mg/L of isoprene from cells expressing a Kudzuisoprene synthase. Expressing a DXS nucleic acid, an IDI nucleic acid,and/or one or more MVA pathway nucleic acids (such as nucleic acidsencoding the entire MVA pathway) in addition to an isoprene synthasenucleic acid may increase the production of isoprene from glycerol.

In some embodiments, an oil is included in the cell medium. For example,B. subtilis cells containing a kudzu isoprene synthase nucleic acidproduced isoprene when cultured in a cell medium containing an oil and asource of glucose (Example 13, part III). In some embodiments, more thanone oil (such as 2, 3, 4, 5, or more oils) is included in the cellmedium. While not intending to be bound to any particular theory, it isbelieved that (i) the oil may increase the amount of carbon in the cellsthat is available for conversion to isoprene, (ii) the oil may increasethe amount of acetyl-CoA in the cells, thereby increasing the carbonflow through the MVA pathway, and/or (ii) the oil may provide extranutrients to the cells, which is desirable since a lot of the carbon inthe cells is converted to isoprene rather than other products. In someembodiments, cells that are cultured in a cell medium containing oilnaturally use the MVA pathway to produce isoprene or are geneticallymodified to contain nucleic acids for the entire MVA pathway. In someembodiments, the oil is partially or completely hydrolyzed before beingadded to the cell culture medium to facilitate the use of the oil by thehost cells.

One of the major hurdles to commercial production of small moleculessuch as isoprene in cells (e.g., bacteria) is the decoupling ofproduction of the molecule from growth of the cells. In some embodimentsfor the commercially viable production of isoprene, a significant amountof the carbon from the feedstock is converted to isoprene, rather thanto the growth and maintenance of the cells (“carbon efficiency”). Invarious embodiments, the cells convert greater than or about 0.0015,0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5,4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture mediuminto isoprene. In particular embodiments, a significant portion of thecarbon from the feedstock that is converted to downstream products isconverted to isoprene. As described further in Example 19, E. coli cellsexpressing MVA pathway and kudzu isoprene synthase nucleic acidsexhibited decoupling of the production of isoprene or the intermediatemevalonic acid from growth, resulting in high carbon efficiency. Inparticular, mevalonic acid was formed from cells expressing the upperMVA pathway from Enterococcus faecalis. Isoprene was formed from cellsexpressing the upper MVA pathway from Enterococcus faecalis, the lowerMVA pathway from Saccharomyces cerevisiae, and the isoprene synthasefrom Pueraria montana (Kudzu). This decoupling of isoprene or mevalonicacid production from growth was demonstrated in four different strainsof E. coli: BL21(LDE3), BL21(LDE3) Tuner, FM5, and MG1655. The first twoE. coli strains are B strains, and the latter two are K12 strains.Decoupling of production from growth was also demonstrated in a variantof MG1655 with ack and pta genes deleted. This variant also demonstratedless production of acetate.

The vast majority of isoprene is derived from petrochemical sources asan impure C5 hydrocarbon fraction which requires extensive purificationbefore the material is suitable for polymerization. Several impuritiesare particularly problematic given their structural similarity toisoprene and the fact that they can act as polymerization catalystpoisons. Such compounds include 1,3-cyclopentadiene,trans-1,3-pentadiene, cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne,and cis-pent-3-ene-1-yne (FIG. 90). In other embodiments, the impuritiescan be 3-hexen-1-ol, 3-hexen-1-yl acetate, limonene, geraniol(trans-3,7-dimethyl-2,6-octadien-1-ol) and citronellol(3,7-dimethyl-6-octen-1-ol). In some embodiments, the isoprenecomposition of the invention is substantially free of any contaminatingunsaturated C5 hydrocarbons. No detectable amount of unsaturated C5hydrocarbons other than isoprene (such as 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne,2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) was found in isoprenecompositions produced using the methods described herein. Some isoprenecompositions produced using the methods described herein containethanol, acetone, and C5 prenyl alcohols as determined by GC/MSanalysis. All of these components are far more readily removed from theisoprene stream than the isomeric C5 hydrocarbon fractions that arepresent in isoprene compositions derived from petrochemical sources.Accordingly, in some embodiments, the isoprene compositions of theinvention require minimal treatment in order to be of polymerizationgrade.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides andnucleic acids can be used in the compositions and methods of theinvention.

As used herein, “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides. In someembodiments, the fusion polypeptide includes part or all of a firstpolypeptide (e.g., an isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide or catalytically active fragment thereof) and may optionallyinclude part or all of a second polypeptide (e.g., a peptide thatfacilitates purification or detection of the fusion polypeptide, such asa His-tag). In some embodiments, the fusion polypeptide has an activityof two or more MVA pathway polypeptides (such as AA-CoA thiolase andHMG-CoA reductase polypeptides). In some embodiments, the polypeptide isa naturally-occurring polypeptide (such as the polypeptide encoded by anEnterococcus faecalis mvaE nucleic acid) that has an activity of two ormore MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100,150, 175, 200, 250, 300, 350, 400, or more amino acids. In someembodiments, the polypeptide fragment contains at least or about 25, 50,75, 100, 150, 200, 300, or more contiguous amino acids from afull-length polypeptide and has at least or about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of acorresponding full-length polypeptide. In particular embodiments, thepolypeptide includes a segment of or the entire amino acid sequence ofany naturally-occurring isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide. In some embodiments, the polypeptide has one or moremutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. As usedherein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

In some embodiments, the polypeptide is a heterologous polypeptide. By“heterologous polypeptide” is meant a polypeptide whose amino acidsequence is not identical to that of another polypeptide naturallyexpressed in the same host cell. In particular, a heterologouspolypeptide is not identical to a wild-type nucleic acid that is foundin the same host cell in nature.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides in either single ordouble-stranded form. In some embodiments, the nucleic acid is arecombinant nucleic acid. By “recombinant nucleic acid” means a nucleicacid of interest that is free of one or more nucleic acids (e.g., genes)which, in the genome occurring in nature of the organism from which thenucleic acid of interest is derived, flank the nucleic acid of interest.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, ora cDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. In various embodiments, a nucleic acidis a recombinant nucleic acid. In some embodiments, an isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid is operably linked toanother nucleic acid encoding all or a portion of another polypeptidesuch that the recombinant nucleic acid encodes a fusion polypeptide thatincludes an isoprene synthase, DXS, IDI, or MVA pathway polypeptide andall or part of another polypeptide (e.g., a peptide that facilitatespurification or detection of the fusion polypeptide, such as a His-tag).In some embodiments, part or all of a recombinant nucleic acid ischemically synthesized. It is to be understood that mutations, includingsingle nucleotide mutations, can occur within a nucleic acid as definedherein.

In some embodiments, the nucleic acid is a heterologous nucleic acid. By“heterologous nucleic acid” is meant a nucleic acid whose nucleic acidsequence is not identical to that of another nucleic acid naturallyfound in the same host cell.

In particular embodiments, the nucleic acid includes a segment of or theentire nucleic acid sequence of any naturally-occurring isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid. In some embodiments,the nucleic acid includes at least or about 50, 100, 150, 200, 300, 400,500, 600, 700, 800, or more contiguous nucleotides from anaturally-occurring isoprene synthase nucleic acid DXS, IDI, or MVApathway nucleic acid. In some embodiments, the nucleic acid has one ormore mutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathway nucleicacid. In some embodiments, the nucleic acid has one or more mutations(e.g., a silent mutation) that increase the transcription or translationof isoprene synthase, DXS, IDI, or MVA pathway nucleic acid. In someembodiments, the nucleic acid is a degenerate variant of any nucleicacid encoding an isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a nucleic acid for improved expression in a host cell, itis desirable in some embodiments to design the nucleic acid such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/orMVA pathway polypeptides and nucleic acids are listed in Appendix 1 (theaccession numbers of Appendix 1 and their corresponding sequences areherein incorporated by reference in their entireties, particularly withrespect to the amino acid and nucleic acid sequences of isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids).The Kegg database also contains the amino acid and nucleic acidsequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVApathway polypeptides and nucleic acids (see, for example, the world-wideweb at “genome.jp/kegg/pathway/map/map00100.html” and the sequencestherein, which are each hereby incorporated by reference in theirentireties, particularly with respect to the amino acid and nucleic acidsequences of isoprene synthase, DXS, IDI, and/or MVA pathwaypolypeptides and nucleic acids). In some embodiments, one or more of theisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/ornucleic acids have a sequence identical to a sequence publicly availableon Dec. 12, 2007 or Sep. 14, 2008, such as any of the sequences thatcorrespond to any of the accession numbers in Appendix 1 or any of thesequences present in the Kegg database. Additional exemplary isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acidsare described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethylallyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Standard methods can be used to determine whethera polypeptide has isoprene synthase polypeptide activity by measuringthe ability of the polypeptide to convert DMAPP into isoprene in vitro,in a cell extract, or in vivo. In an exemplary assay, cell extracts areprepared by growing a strain (e.g., the E. coli/pTrcKudzu straindescribed herein) in the shake flask method as described in Example 10.After induction is complete, approximately 10 mL of cells are pelletedby centrifugation at 7000×g for 10 minutes and resuspended in 5 ml ofPEB without glycerol. The cells are lysed using a French Pressure cellusing standard procedures. Alternatively the cells are treated withlysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thawat −80 C.

Isoprene synthase polypeptide activity in the cell extract can bemeasured, for example, as described in Silver et al., J. Biol. Chem.270:13010-13016, 1995 and references therein, which are each herebyincorporated by reference in their entireties, particularly with respectto assays for isoprene synthase polypeptide activity. DMAPP (Sigma) isevaporated to dryness under a stream of nitrogen and rehydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μL of 1M MgCl₂,1 mM (250 μg/ml) DMAPP, 65 μL of Plant Extract Buffer (PEB) (50 mMTris-HCl, pH 8.0, 20 mM MgCl₂, 5% glycerol, and 2 mM DTT) is added to 25μL of cell extract in a 20 ml Headspace vial with a metal screw cap andteflon coated silicon septum (Agilent Technologies) and cultured at 37°C. for 15 minutes with shaking. The reaction is quenched by adding 200μL of 250 mM EDTA and quantified by GC/MS as described in Example 10,part II.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Exemplary isoprene synthase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acidis from the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the isoprene synthase polypeptide or nucleic acid is apolypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey etal., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (suchas Populus alba, Populus nigra, Populus trichocarpa, or Populusalba×tremula (CAC35696) Miller et al., Planta 213: 483-487, 2001) aspen(such as Populus tremuloides) Silver et al., JBC 270(22): 13010-1316,1995), or English Oak (Quercus robur) (Zimmer et al., WO 98/02550),which are each hereby incorporated by reference in their entireties,particularly with respect to isoprene synthase nucleic acids and theexpression of isoprene synthase polypeptides. Suitable isoprenesynthases include, but are not limited to, those identified by GenbankAccession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241,which are each hereby incorporated by reference in their entireties,particularly with respect to sequences of isoprene synthase nucleicacids and polypeptides. In some embodiments, the isoprene synthasepolypeptide or nucleic acid is not a naturally-occurring polypeptide ornucleic acid from Quercus robur (i.e., the isoprene synthase polypeptideor nucleic acid is an isoprene synthase polypeptide or nucleic acidother than a naturally-occurring polypeptide or nucleic acid fromQuercus robur). In some embodiments, the isoprene synthase nucleic acidor polypeptide is a naturally-occurring polypeptide or nucleic acid frompoplar. In some embodiments, the isoprene synthase nucleic acid orpolypeptide is not a naturally-occurring polypeptide or nucleic acidfrom poplar.

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS)polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of a DXS polypeptide.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has DXS polypeptide activity bymeasuring the ability of the polypeptide to convert pyruvate andD-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate invitro, in a cell extract, or in vivo. Exemplary DXS nucleic acidsinclude nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXS polypeptide. Exemplary DXS polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyses the interconversion of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase(AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoAreductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK)polypeptides, phosphomevalonate kinase (PMK) polypeptides,diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonatedecarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK)polypeptides, IDI polypeptides, and polypeptides (e.g., fusionpolypeptides) having an activity of two or more MVA pathwaypolypeptides. In particular, MVA pathway polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway nucleic acids include nucleic acidsthat encode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoAthiolase or AACT) convert two molecules of acetyl-CoA intoacetoacetyl-CoA. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has AA-CoA thiolasepolypeptide activity by measuring the ability of the polypeptide toconvert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in acell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase or HMGS)polypeptides convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as thosedescribed herein) can be used to determine whether a polypeptide hasHMG-CoA synthase polypeptide activity by measuring the ability of thepolypeptide to convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR)polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has HMG-CoA reductase polypeptideactivity by measuring the ability of the polypeptide to convert3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cellextract, or in vivo.

Mevalonate kinase (MVK) polypeptides phosphorylates mevalonate to formmevalonate-5-phosphate. Standard methods (such as those describedherein) can be used to determine whether a polypeptide has MVKpolypeptide activity by measuring the ability of the polypeptide toconvert mevalonate into mevalonate-5-phosphate in vitro, in a cellextract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylatesmevalonate-5-phosphate to form mevalonate-5-diphosphate. Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has PMK polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-phosphate intomevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonate decarboxylase (MVD or DPMDC) polypeptides convertmevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has MVD polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-diphosphate into IPPin vitro, in a cell extract, or in vivo.

Phosphomevalonate decarboxylase (PMDC) polypeptides convertmevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has PMDC polypeptide activity by measuring the ability ofthe polypeptide to convert mevalonate-5-phosphate into IP in vitro, in acell extract, or in vivo.

Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentylphosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has IPK polypeptide activity by measuring the ability of thepolypeptide to convert IP into IPP in vitro, in a cell extract, or invivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids can beisolated using standard methods. Methods of obtaining desired nucleicacids from a source organism of interest (such as a bacterial genome)are common and well known in the art of molecular biology (see, forexample, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to the isolation of nucleic acids of interest). For example, ifthe sequence of the nucleic acid is known (such as any of the knownnucleic acids described herein), suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired nucleic acid sequence. Once thesequence is isolated, the DNA may be amplified using standard primerdirected amplification methods such as polymerase chain reaction (PCR)(U.S. Pat. No. 4,683,202, which is incorporated by reference in itsentirety, particularly with respect to PCR methods) to obtain amounts ofDNA suitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacids (such as any isoprene synthase, DXS, IDI, and/or MVA pathwaynucleic acids with a known nucleic acid sequence) can be chemicallysynthesized using standard methods.

Additional isoprene synthase, DXS, IDI, or MVA pathway polypeptides andnucleic acids which may be suitable for use in the compositions andmethods described herein can be identified using standard methods. Forexample, cosmid libraries of the chromosomal DNA of organisms known toproduce isoprene naturally can be constructed in organisms such as E.coli, and then screened for isoprene production. In particular, cosmidlibraries may be created where large segments of genomic DNA (35-45 kb)are packaged into vectors and used to transform appropriate hosts.Cosmid vectors are unique in being able to accommodate large quantitiesof DNA. Generally cosmid vectors have at least one copy of the cos DNAsequence which is needed for packaging and subsequent circularization ofthe heterologous DNA. In addition to the cos sequence, these vectorsalso contain an origin of replication such as ColEI and drug resistancemarkers such as a nucleic acid resistant to ampicillin or neomycin.Methods of using cosmid vectors for the transformation of suitablebacterial hosts are well described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989,which is hereby incorporated by reference in its entirety, particularlywith respect to transformation methods.

Typically to clone cosmids, heterologous DNA is isolated using theappropriate restriction endonucleases and ligated adjacent to the cosregion of the cosmid vector using the appropriate ligases. Cosmidvectors containing the linearized heterologous DNA are then reacted witha DNA packaging vehicle such as bacteriophage. During the packagingprocess, the cos sites are cleaved and the heterologous DNA is packagedinto the head portion of the bacterial viral particle. These particlesare then used to transfect suitable host cells such as E. coli. Onceinjected into the cell, the heterologous DNA circularizes under theinfluence of the cos sticky ends. In this manner, large segments ofheterologous DNA can be introduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, and/or MVApathway nucleic acids include screening a metagenomic library by assay(such as the headspace assay described herein) or by PCR using primersdirected against nucleotides encoding for a length of conserved aminoacids (for example, at least 3 conserved amino acids). Conserved aminoacids can be identified by aligning amino acid sequences of knownisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides. Conservedamino acids for isoprene synthase polypeptides can be identified basedon aligned sequences of known isoprene synthase polypeptides. Anorganism found to produce isoprene naturally can be subjected tostandard protein purification methods (which are well known in the art)and the resulting purified polypeptide can be sequenced using standardmethods. Other methods are found in the literature (see, for example,Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007;Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which areeach hereby incorporated by reference in their entireties, particularlywith respect to identification of nucleic acids involved in thesynthesis of isoprene).

Additionally, standard sequence alignment and/or structure predictionprograms can be used to identify additional DXS, IDI, or MVA pathwaypolypeptides and nucleic acids based on the similarity of their primaryand/or predicted polypeptide secondary structure with that of known DXS,IDI, or MVA pathway polypeptides and nucleic acids. Standard databasessuch as the swissprot-trembl database (world-wide web at “expasy.org”,Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue MichelServet CH-1211 Geneva 4, Switzerland) can also be used to identifyisoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleicacids. The secondary and/or tertiary structure of an isoprene synthase,DXS, IDI, or MVA pathway polypeptide can be predicted using the defaultsettings of standard structure prediction programs, such asPredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA).Alternatively, the actual secondary and/or tertiary structure of anisoprene synthase, DXS, IDI, or MVA pathway polypeptide can bedetermined using standard methods. Additional isoprene synthase, DXS,IDI, or MVA pathway nucleic acids can also be identified byhybridization to probes generated from known isoprene synthase, DXS,IDI, or MVA pathway nucleic acids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, or MVA pathway nucleic aciddescribed herein can be included in one or more vectors. Accordingly,the invention also features vectors with one more nucleic acids encodingany of the isoprene synthase, DXS, IDI, or MVA pathway polypeptides thatare described herein. As used herein, a “vector” means a construct thatis capable of delivering, and desirably expressing one or more nucleicacids of interest in a host cell. Examples of vectors include, but arenot limited to, plasmids, viral vectors, DNA or RNA expression vectors,cosmids, and phage vectors. In some embodiments, the vector contains anucleic acid under the control of an expression control sequence.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An “inducible promoter” is apromoter that is active under environmental or developmental regulation.The expression control sequence is operably linked to the nucleic acidsegment to be transcribed.

In some embodiments, the vector contains a selective marker. The term“selective marker” refers to a nucleic acid capable of expression in ahost cell that allows for ease of selection of those host cellscontaining an introduced nucleic acid or vector. Examples of selectablemarkers include, but are not limited to, antibiotic resistance nucleicacids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin,hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/ornucleic acids that confer a metabolic advantage, such as a nutritionaladvantage on the host cell. Exemplary nutritional selective markersinclude those markers known in the art as amdS, argB, and pyr4. Markersuseful in vector systems for transformation of Trichoderma are known inthe art (see, e.g., Finkelstein, Chapter 6 in Biotechnology ofFilamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann,Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied MolecularGenetics of Filamentous Fungi, Blackie Academic and Professional,Chapman and Hall, London, 1992, which are each hereby incorporated byreference in their entireties, particularly with respect to selectivemarkers). In some embodiments, the selective marker is the amdS nucleicacid, which encodes the enzyme acetamidase, allowing transformed cellsto grow on acetamide as a nitrogen source. The use of an A. nidulansamdS nucleic acid as a selective marker is described in Kelley et al.,EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987(which are each hereby incorporated by reference in their entireties,particularly with respect to selective markers). In some embodiments, anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid integrates intoa chromosome of the cells without a selective marker.

Suitable vectors are those which are compatible with the host cellemployed. Suitable vectors can be derived, for example, from abacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), acosmid, a yeast, or a plant. Protocols for obtaining and using suchvectors are known to those in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, 1989, which is hereby incorporated by reference in its entirety,particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in thehost cell can be used for expression of an isoprene synthase, DXS, IDI,or MVA pathway nucleic acid in the host cell. Initiation control regionsor promoters, which are useful to drive expression of isoprene synthase,DXS, IDI, or MVA pathway nucleic acids in various host cells arenumerous and familiar to those skilled in the art (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors for the expression of nucleic acids of interest). Virtuallyany promoter capable of driving these nucleic acids is suitable for thepresent invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI(useful for expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, trp, λP_(L), λP_(R), T7, tac, and trc (useful forexpression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, forexample, U.S. Pat. No. 7,132,527 and references cited therein, which areeach hereby incorporated by reference in their entireties, particularlywith respect promoters and plasmid systems for expressing polypeptidesof interest). Reported glucose isomerase promoter mutants can be used tovary the level of expression of the polypeptide encoded by a nucleicacid operably linked to the glucose isomerase promoter (U.S. Pat. No.7,132,527). In various embodiments, the glucose isomerase promoter iscontained in a low, medium, or high copy plasmid (U.S. Pat. No.7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, and/or MVApathway nucleic acid is contained in a low copy plasmid (e.g., a plasmidthat is maintained at about 1 to about 4 copies per cell), medium copyplasmid (e.g., a plasmid that is maintained at about 10 to about 15copies per cell), or high copy plasmid (e.g., a plasmid that ismaintained at about 50 or more copies per cell). In some embodiments,the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a T7 promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a T7 promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Trc promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Trc promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Lac promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Lac promoteris contained in a low copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to an endogenous promoter, suchas an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces,or Trichoderma promoter or an endogenous alkaline serine protease,isoprene synthase, DXS, IDI, or MVA pathway promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to an endogenouspromoter is contained in a high copy plasmid. In some embodiments, thevector is a replicating plasmid that does not integrate into achromosome in the cells. In some embodiments, part or all of the vectorintegrates into a chromosome in the cells.

In some embodiments, the vector is any vector which when introduced intoa fungal host cell is integrated into the host cell genome and isreplicated. Reference is made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, the world-wide web at “fgsc.net” and thereferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to vectors) fora list of vectors. Additional examples of suitable expression and/orintegration vectors are provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987,Supplement 30, section 7.7.18); van den Hondel et al. in Bennett andLasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp.396-428, 1991; and U.S. Pat. No. 5,874,276, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors. Particularly useful vectors include pFB6, pBR322, PUC18,pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid is operably linked to a suitable promoter that showstranscriptional activity in a fungal host cell. The promoter may bederived from one or more nucleic acids encoding a polypeptide that iseither endogenous or heterologous to the host cell. In some embodiments,the promoter is useful in a Trichoderma host. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2,xyn1, and amy. In some embodiments, the promoter is one that is nativeto the host cell. For example, in some embodiments when T. reesei is thehost, the promoter is a native T. reesei promoter. In some embodiments,the promoter is T. reesei cbh1, which is an inducible promoter and hasbeen deposited in GenBank under Accession No. D86235, which isincorporated by reference in its entirety, particularly with respect topromoters. In some embodiments, the promoter is one that is heterologousto the fungal host cell. Other examples of useful promoters includepromoters from the genes of A. awamori and A. niger glucoamylase (glaA)(Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et al., EMBOJ. 3:1581-1585, 1984, which are each hereby incorporated by reference intheir entireties, particularly with respect to promoters); Aspergillusniger alpha amylases, Aspergillus oryzae TAKA amylase, T. reesei xln1,and the T. reesei cellobiohydrolase 1 (EP 137280, which is incorporatedby reference in its entirety, particularly with respect to promoters).

In some embodiments, the expression vector also includes a terminationsequence. Termination control regions may also be derived from variousgenes native to the host cell. In some embodiments, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is endogenous to the hostcell. A particularly suitable terminator sequence is cbh1 derived from aTrichoderma strain (such as T. reesei). Other useful fungal terminatorsinclude the terminator from an A. niger or A. awamori glucoamylasenucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boelet al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated byreference in their entireties, particularly with respect to fungalterminators). Optionally, a termination site may be included. Foreffective expression of the polypeptides, DNA encoding the polypeptideare linked operably through initiation codons to selected expressioncontrol regions such that expression results in the formation of theappropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator alloriginate from the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid to be expressed. In some embodiments, the coding region for anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid is insertedinto a general-purpose expression vector such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, or MVA pathway nucleic acid can beincorporated into a vector, such as an expression vector, using standardtechniques (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982, which is hereby incorporated by reference inits entirety, particularly with respect to the screening of appropriateDNA sequences and the construction of vectors). Methods used to ligatethe DNA construct comprising a nucleic acid of interest (such as anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid), a promoter, aterminator, and other sequences and to insert them into a suitablevector are well known in the art. For example, restriction enzymes canbe used to cleave the isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid and the vector. Then, the compatible ends of the cleavedisoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the cleavedvector can be ligated. Linking is generally accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide linkers are used in accordance with conventionalpractice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More GeneManipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to oligonucleotide linkers). Additionally,vectors can be constructed using known recombination techniques (e.g.,Invitrogen Life Technologies, Gateway Technology).

In some embodiments, it may be desirable to over-express isoprenesynthase, DXS, IDI, or MVA pathway nucleic acids at levels far higherthan currently found in naturally-occurring cells. This result may beaccomplished by the selective cloning of the nucleic acids encodingthose polypeptides into multicopy plasmids or placing those nucleicacids under a strong inducible or constitutive promoter. Methods forover-expressing desired polypeptides are common and well known in theart of molecular biology and examples may be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, which is hereby incorporated by reference in its entirety,particularly with respect to cloning techniques.

The following resources include descriptions of additional generalmethodology useful in accordance with the invention: Kreigler, GeneTransfer and Expression; A Laboratory Manual, 1990 and Ausubel et al.,Eds. Current Protocols in Molecular Biology, 1994, which are each herebyincorporated by reference in their entireties, particularly with respectto molecular biology and cloning techniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, or MVA pathway nucleic acids (and theirencoded polypeptides) can be obtained from any organism that naturallycontains isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids.As noted above, isoprene is formed naturally by a variety of organisms,such as bacteria, yeast, plants, and animals. Organisms contain the MVApathway, DXP pathway, or both the MVA and DXP pathways for producingisoprene (FIGS. 19A and 19B). Thus, DXS nucleic acids can be obtained,e.g., from any organism that contains the DXP pathway or contains boththe MVA and DXP pathways. IDI and isoprene synthase nucleic acids can beobtained, e.g., from any organism that contains the MVA pathway, DXPpathway, or both the MVA and DXP pathways. MVA pathway nucleic acids canbe obtained, e.g., from any organism that contains the MVA pathway orcontains both the MVA and DXP pathways.

In some embodiments, the nucleic acid sequence of the isoprene synthase,DXS, IDI, or MVA pathway nucleic is identical to the sequence of anucleic acid that is produced by any of the following organisms innature. In some embodiments, the amino acid sequence of the isoprenesynthase, DXS, IDI, or MVA pathway polypeptide is identical to thesequence of a polypeptide that is produced by any of the followingorganisms in nature. In some embodiments, the isoprene synthase, DXS,IDI, or MVA pathway nucleic acid or polypeptide is a mutant nucleic acidor polypeptide derived from any of the organisms described herein. Asused herein, “derived from” refers to the source of the nucleic acid orpolypeptide into which one or more mutations is introduced. For example,a polypeptide that is “derived from a plant polypeptide” refers topolypeptide of interest that results from introducing one or moremutations into the sequence of a wild-type (i.e., a sequence occurringin nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of whichare species of Aspergillus such as A. oryzae and A. niger, species ofSaccharomyces such as S. cerevisiae, species of Schizosaccharomyces suchas S. pombe, and species of Trichoderma such as T. reesei. In someembodiments, the source organism is a filamentous fungal cell. The term“filamentous fungi” refers to all filamentous forms of the subdivisionEumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,Wiley, New York). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligatory aerobic. The filamentous fungal parent cell may be a cell ofa species of, but not limited to, Trichoderma, (e.g., Trichodermareesei, the asexual morph of Hypocrea jecorina, previously classified asT. longibrachiatum, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp.,Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea);Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillussp. (e.g., A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, orA. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993and Goedegebuur et al., Genet 41: 89-98, 2002), Fusarium sp., (e.g., F.roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum),Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M.miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci.228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or“Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae,A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F.oxysporum, or F. solani. Aspergillus strains are disclosed in Ward etal., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur etal., Curr Gene 41:89-98, 2002, which are each hereby incorporated byreference in their entireties, particularly with respect to fungi. Inparticular embodiments, the fungus is a strain of Trichoderma, such as astrain of T. reesei. Strains of T. reesei are known and non-limitingexamples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCCNo. 56765, ATCC No. 56767, and NRRL 15709, which are each herebyincorporated by reference in their entireties, particularly with respectto strains of T. reesei. In some embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated byreference in its entirety, particularly with respect to strains of T.reesei.

In some embodiments, the source organism is a yeast, such asSaccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.

In some embodiments, the source organism is a bacterium, such as strainsof Bacillus such as B. lichenformis or B. subtilis, strains of Pantoeasuch as P. citrea, strains of Pseudomonas such as P. alcaligenes,strains of Streptomyces such as S. lividans or S. rubiginosus, orstrains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium.Non-limiting examples include strains of Streptomyces (e.g., S.lividans, S. coelicolor, or S. griseus) and Bacillus. In someembodiments, the source organism is a gram-negative bacterium, such asE. coli or Pseudomonas sp.

In some embodiments, the source organism is a plant, such as a plantfrom the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the source organism is kudzu, poplar (such as Populusalba×tremula CAC35696), aspen (such as Populus tremuloides), or Quercusrobur.

In some embodiments, the source organism is an algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such ascyanobacteria classified into any of the following groups based onmorphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales,or Stigonematales.

In some embodiments, the source organism is an archaeon, such asMethanosarcina mazei. Exemplary archaea include those disclosed by Kogaand Morii (Microbiology & Mol. Biology Reviews, 71:97-120, 2007, whichis hereby incorporated by reference in its entirety, particularly withrespect to archaea (see Table 3)). Other exemplary archaea arehyperthermophilic archaea, such as Methanococcus jannaschii (Huang etal., Protein Expression and Purification 17(1):33-40, 1999) andhalophilic archaea (such as Halobacterium salanarium).

TABLE 3 Exemplary archaea Exemplary Original name Strain Name mostrecently proposed Caldariella acidophila Sulfolobus solfataricusHalobacterium cutirubrum Halobacterium salinarum Halobacterium halobiumHalobacterium salinarum Halobacterium mediterranei Haloferaxmediterranei Halobacterium vallismortis Haloarcula vallismortisMethanobacterium ΔH Methanothermobacter thermoautotrophicumthermautotrophicus Methanobacterium Marburg Methanothermobacterthermoautotrophicum marburgensis Methanobacterium SF-4Methanothermobacter wolfeii thermoformicicum Methanococcus igneusMethanotorris igneus Natronobacterium pharaonis Natronomonas pharaonisPseudomonas salinaria Halobacterium salinarumExemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS,IDI, and/or MVA pathway polypeptides and to produce isoprene in themethods of the invention. Exemplary host cells include cells from any ofthe organisms listed in the prior section under the heading “ExemplarySource Organisms.” The host cell may be a cell that naturally producesisoprene or a cell that does not naturally produce isoprene. In someembodiments, the host cell naturally produces isoprene using the DXPpathway, and an isoprene synthase, DXS, and/or IDI nucleic acid is addedto enhance production of isoprene using this pathway. In someembodiments, the host cell naturally produces isoprene using the MVApathway, and an isoprene synthase and/or one or more MVA pathway nucleicacids are added to enhance production of isoprene using this pathway. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway and one or more MVA pathway nucleic acids are added toproduce isoprene using part or all of the MVA pathway as well as the DXPpathway. In some embodiments, the host cell naturally produces isopreneusing both the DXP and MVA pathways and one or more isoprene synthase,DXS, IDI, or MVA pathway nucleic acids are added to enhance productionof isoprene by one or both of these pathways.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids or vectorscontaining them can be inserted into a host cell (e.g., a plant cell, afungal cell, a yeast cell, or a bacterial cell described herein) usingstandard techniques for expression of the encoded isoprene synthase,DXS, IDI, and/or MVA pathway polypeptide. Introduction of a DNAconstruct or vector into a host cell can be performed using techniquessuch as transformation, electroporation, nuclear microinjection,transduction, transfection (e.g., lipofection mediated or DEAE-Dextrinmediated transfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (see, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods). The expression of heterologous polypeptide in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; U.S. Pat.No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme Microb. Technol.13:227-233, 1991; Harkki et al., Bio Technol. 7:596-603, 1989; EP244,234; EP 215,594; and Nevalainen et al., “The Molecular Biology ofTrichoderma and its Application to the Expression of Both Homologous andHeterologous Genes,” in Molecular Industrial Mycology, Eds. Leong andBerka, Marcel Dekker Inc., NY pp. 129-148, 1992, which are each herebyincorporated by reference in their entireties, particularly with respectto transformation and expression methods). Reference is also made to Caoet al., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al.,Proceedings. Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are eachhereby incorporated by reference in their entireties, particularly withrespect to transformation methods) for transformation of Aspergillusstrains. The introduced nucleic acids may be integrated into chromosomalDNA or maintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In onenon-limiting example, stable transformants including an amdS marker aredistinguished from unstable transformants by their faster growth rateand the formation of circular colonies with a smooth, rather than raggedoutline on solid culture medium containing acetamide. Additionally, insome cases a further test of stability is conducted by growing thetransformants on a solid non-selective medium (e.g., a medium that lacksacetamide), harvesting spores from this culture medium, and determiningthe percentage of these spores which subsequently germinate and grow onselective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a known manner. In one specificembodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia (see,Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated byreference in its entirety, particularly with respect to transformationmethods). In some embodiments, the mycelia are obtained from germinatedvegetative spores. The mycelia are treated with an enzyme that digeststhe cell wall resulting in protoplasts. The protoplasts are thenprotected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M. It is desirable to useabout a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally, between about 10 mM CaCl₂ and 50mM CaCl₂ is used in an uptake solution. In addition to the calcium ionin the uptake solution, other compounds generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). While not intending to be bound to any particular theory,it is believed that the polyethylene glycol acts to fuse the cellmembranes, thus permitting the contents of the medium to be deliveredinto the cytoplasm of the Trichoderma sp. strain and the plasmid DNA tobe transferred to the nucleus. This fusion frequently leaves multiplecopies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA.Generally, a high concentration of PEG is added to the uptake solution.From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplastsuspension. In some embodiments, about 0.25 volumes are added to theprotoplast suspension. Additives such as dimethyl sulfoxide, heparin,spermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation. Similar procedures areavailable for other fungal host cells (see, e.g., U.S. Pat. Nos.6,022,725 and 6,268,328, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods).

Generally, the mixture is then cultured at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired nucleic acidsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is desirably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then cultured either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. When the growth medium includes a growth selection (e.g.,acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according toconventional methods, e.g., as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is herebyincorporated by reference in its entirety, particularly with respect totransformation methods.

Exemplary Cell Culture Media

The invention also includes a cell or a population of cells in culturethat produce isoprene. By “cells in culture” is meant two or more cellsin a solution (e.g., a cell medium) that allows the cells to undergo oneor more cell divisions. “Cells in culture” do not include plant cellsthat are part of a living, multicellular plant containing cells thathave differentiated into plant tissues. In various embodiments, the cellculture includes at least or about 10, 20, 50, 100, 200, 500, 1,000,5,000, 10,000 or more cells.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells may include any carbonsource suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharide),invert sugar (e.g., enzymatically treated sucrose syrup), glycerol,glycerine (e.g., a glycerine byproduct of a biodiesel or soap-makingprocess), dihydroxyacetone, one-carbon source, oil (e.g., a plant orvegetable oil such as corn, palm, or soybean oil), animal fat, animaloil, fatty acid (e.g., a saturated fatty acid, unsaturated fatty acid,or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbialor plant protein or peptide), renewable carbon source (e.g., a biomasscarbon source such as a hydrolyzed biomass carbon source), yeastextract, component from a yeast extract, polymer, acid, alcohol,aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, orany combination of two or more of the foregoing. In some embodiments,the carbon source is a product of photosynthesis, including, but notlimited to, glucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose). In some embodiments, the cellmedium includes a carbohydrate as well as a carbon source other than acarbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, or a component from a yeast extract). In some embodiments, thecell medium includes a carbohydrate as well as a polypeptide (e.g., amicrobial or plant protein or peptide). In some embodiments, themicrobial polypeptide is a polypeptide from yeast or bacteria. In someembodiments, the plant polypeptide is a polypeptide from soy, corn,canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at leastor about 5 grams per liter of broth (g/L, wherein the volume of brothincludes both the volume of the cell medium and the volume of thecells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100,150, 200, 300, 400, or more g/L. In some embodiments, the concentrationof the carbohydrate is between about 50 and about 400 g/L, such asbetween about 100 and about 360 g/L, between about 120 and about 360g/L, or between about 200 and about 300 g/L. In some embodiments, thisconcentration of carbohydrate includes the total amount of carbohydratethat is added before and/or during the culturing of the host cells.

In some embodiments, the cells are cultured under limited glucoseconditions. By “limited glucose conditions” is meant that the amount ofglucose that is added is less than or about 105% (such as about 100%) ofthe amount of glucose that is consumed by the cells. In particularembodiments, the amount of glucose that is added to the culture mediumis approximately the same as the amount of glucose that is consumed bythe cells during a specific period of time. In some embodiments, therate of cell growth is controlled by limiting the amount of addedglucose such that the cells grow at the rate that can be supported bythe amount of glucose in the cell medium. In some embodiments, glucosedoes not accumulate during the time the cells are cultured. In variousembodiments, the cells are cultured under limited glucose conditions forgreater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or70 hours. In various embodiments, the cells are cultured under limitedglucose conditions for greater than or about 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time thecells are cultured. While not intending to be bound by any particulartheory, it is believed that limited glucose conditions may allow morefavorable regulation of the cells.

In some embodiments, the cells are cultured in the presence of an excessof glucose. In particular embodiments, the amount of glucose that isadded is greater than about 105% (such as about or greater than 110,120, 150, 175, 200, 250, 300, 400, or 500%) or more of the amount ofglucose that is consumed by the cells during a specific period of time.In some embodiments, glucose accumulates during the time the cells arecultured.

Exemplary lipids are any substance containing one or more fatty acidsthat are C4 and above fatty acids that are saturated, unsaturated, orbranched.

Exemplary oils are lipids that are liquid at room temperature. In someembodiments, the lipid contains one or more C4 or above fatty acids(e.g., contains one or more saturated, unsaturated, or branched fattyacid with four or more carbons). In some embodiments, the oil isobtained from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, linseed, oleagineous microbial cells, Chinese tallow, or anycombination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R”is a hydrocarbon. Exemplary unsaturated fatty acids include compoundswhere “R” includes at least one carbon-carbon double bond. Exemplaryunsaturated fatty acids include, but are not limited to, oleic acid,vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.Exemplary polyunsaturated fatty acids include compounds where “R”includes a plurality of carbon-carbon double bonds. Exemplary saturatedfatty acids include compounds where “R” is a saturated aliphatic group.In some embodiments, the carbon source includes one or more C₁₂-C₂₂fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fattyacid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplaryembodiment, the fatty acid is palmitic acid. In some embodiments, thecarbon source is a salt of a fatty acid (e.g., an unsaturated fattyacid), a derivative of a fatty acid (e.g., an unsaturated fatty acid),or a salt of a derivative of fatty acid (e.g., an unsaturated fattyacid). Suitable salts include, but are not limited to, lithium salts,potassium salts, sodium salts, and the like. Di- and triglycerols arefatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fattyacid, monoglyceride, diglyceride, or triglyceride is at least or about 1gram per liter of broth (g/L, wherein the volume of broth includes boththe volume of the cell medium and the volume of the cells), such as atleast or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300,400, or more g/L. In some embodiments, the concentration of the lipid,oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride isbetween about 10 and about 400 g/L, such as between about 25 and about300 g/L, between about 60 and about 180 g/L, or between about 75 andabout 150 g/L. In some embodiments, the concentration includes the totalamount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride,or triglyceride that is added before and/or during the culturing of thehost cells. In some embodiments, the carbon source includes both (i) alipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglycerideand (ii) a carbohydrate, such as glucose. In some embodiments, the ratioof the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, ortriglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e.,one carbon in the lipid, oil, fat, fatty acid, monoglyceride,diglyceride, or triglyceride per carbohydrate carbon). In particularembodiments, the amount of the lipid, oil, fat, fatty acid,monoglyceride, diglyceride, or triglyceride is between about 60 and 180g/L, and the amount of the carbohydrate is between about 120 and 360g/L.

Exemplary microbial polypeptide carbon sources include one or morepolypeptides from yeast or bacteria. Exemplary plant polypeptide carbonsources include one or more polypeptides from soy, corn, canola,jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt, and components fromany of the foregoing. Exemplary renewable carbon sources also includeglucose, hexose, pentose and xylose present in biomass, such as corn,switchgrass, sugar cane, cell waste of fermentation processes, andprotein by-product from the milling of soy, corn, or wheat. In someembodiments, the biomass carbon source is a lignocellulosic,hemicellulosic, or cellulosic material such as, but are not limited to,a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft woodpulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, cornstover, switch grass, rice hull product, or a by-product from wet or drymilling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat,and/or distillers grains). Exemplary cellulosic materials include wood,paper and pulp waste, herbaceous plants, and fruit pulp. In someembodiments, the carbon source includes any plant part, such as stems,grains, roots, or tubers. In some embodiments, all or part of any of thefollowing plants are used as a carbon source: corn, wheat, rye, sorghum,triticate, rice, millet, barley, cassava, legumes, such as beans andpeas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. Insome embodiments, the carbon source is a biomass hydrolysate, such as abiomass hydrolysate that includes both xylose and glucose or thatincludes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) ispretreated before it is added to the cell culture medium. In someembodiments, the pretreatment includes enzymatic pretreatment, chemicalpretreatment, or a combination of both enzymatic and chemicalpretreatment (see, for example, Farzaneh et al., Bioresource Technology96 (18): 2014-2018, 2005; U.S. Pat. No. 6,176,176; U.S. Pat. No.6,106,888; which are each hereby incorporated by reference in theirentireties, particularly with respect to the pretreatment of renewablecarbon sources). In some embodiments, the renewable carbon source ispartially or completely hydrolyzed before it is added to the cellculture medium.

In some embodiments, the renewable carbon source (such as corn stover)undergoes ammonia fiber expansion (AFEX) pretreatment before it is addedto the cell culture medium (see, for example, Farzaneh et al.,Bioresource Technology 96 (18): 2014-2018, 2005). During AFEXpretreatment, a renewable carbon source is treated with liquid anhydrousammonia at moderate temperatures (such as about 60 to about 100° C.) andhigh pressure (such as about 250 to about 300 psi) for about 5 minutes.Then, the pressure is rapidly released. In this process, the combinedchemical and physical effects of lignin solubilization, hemicellulosehydrolysis, cellulose decrystallization, and increased surface areaenables near complete enzymatic conversion of cellulose andhemicellulose to fermentable sugars. AFEX pretreatment has the advantagethat nearly all of the ammonia can be recovered and reused, while theremaining serves as nitrogen source for microbes in downstreamprocesses. Also, a wash stream is not required for AFEX pretreatment.Thus, dry matter recovery following the AFEX treatment is essentially100%. AFEX is basically a dry to dry process. The treated renewablecarbon source is stable for long periods and can be fed at very highsolid loadings in enzymatic hydrolysis or fermentation processes.Cellulose and hemicellulose are well preserved in the AFEX process, withlittle or no degradation. There is no need for neutralization prior tothe enzymatic hydrolysis of a renewable carbon source that has undergoneAFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sourcesproduces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., arenewable carbon source) is equivalent to at least or about 0.1, 0.5, 1,1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalentamount of glucose can be determined by using standard HPLC methods withglucose as a reference to measure the amount of glucose generated fromthe carbon source. In some embodiments, the concentration of the carbonsource (e.g., a renewable carbon source) is equivalent to between about0.1 and about 20% glucose, such as between about 0.1 and about 10%glucose, between about 0.5 and about 10% glucose, between about 1 andabout 10% glucose, between about 1 and about 5% glucose, or betweenabout 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one ormore components of yeast extract. In some embodiments, the concentrationof yeast extract is at least 1 gram of yeast extract per liter of broth(g/L, wherein the volume of broth includes both the volume of the cellmedium and the volume of the cells), such at least or about 5, 10, 15,20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In someembodiments, the concentration of yeast extract is between about 1 andabout 300 g/L, such as between about 1 and about 200 g/L, between about5 and about 200 g/L, between about 5 and about 100 g/L, or between about5 and about 60 g/L. In some embodiments, the concentration includes thetotal amount of yeast extract that is added before and/or during theculturing of the host cells. In some embodiments, the carbon sourceincludes both yeast extract (or one or more components thereof) andanother carbon source, such as glucose. In some embodiments, the ratioof yeast extract to the other carbon source is about 1:5, about 1:10, orabout 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such ascarbon dioxide, or methanol. Glycerol production from single carbonsources (e.g., methanol, formaldehyde, or formate) has been reported inmethylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543,1989, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources) and in bacteria (Hunter et.al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).These organisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York, 1986, which ishereby incorporated by reference in its entirety, particularly withrespect to carbon sources). The ribulose monophosphate pathway involvesthe condensation of formate with ribulose-5-phosphate to form a sixcarbon sugar that becomes fructose and eventually the three carbonproduct glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed.,415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK,1993, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources). Similarly, various speciesof Candida metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-9, 1990, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containingphysiological salts and nutrients (see, e.g., Pourquie, J. et al.,Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al.,Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ.Microbiol. 63:1298-1306, 1997, which are each hereby incorporated byreference in their entireties, particularly with respect to cellmedias). Exemplary growth media are common commercially prepared mediasuch as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, orYeast medium (YM) broth. Other defined or synthetic growth media mayalso be used, and the appropriate medium for growth of particular hostcells are known by someone skilled in the art of microbiology orfermentation science.

In addition to an appropriate carbon source, the cell medium desirablycontains suitable minerals, salts, cofactors, buffers, and othercomponents known to those skilled in the art suitable for the growth ofthe cultures or the enhancement of isoprene production (see, forexample, WO 2004/033646 and references cited therein and WO 96/35796 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect cell medias andcell culture conditions). In some embodiments where an isoprenesynthase, DXS, IDI, and/or MVA pathway nucleic acid is under the controlof an inducible promoter, the inducing agent (e.g., a sugar, metal saltor antimicrobial), is desirably added to the medium at a concentrationeffective to induce expression of an isoprene synthase, DXS, IDI, and/orMVA pathway polypeptide. In some embodiments, cell medium has anantibiotic (such as kanamycin) that corresponds to the antibioticresistance nucleic acid (such as a kanamycin resistance nucleic acid) ona vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques. In some embodiments, the cellsare cultured in a culture medium under conditions permitting theexpression of one or more isoprene synthase, DXS, IDI, or MVA pathwaypolypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to cell culture and fermentation conditions). Cells are grownand maintained at an appropriate temperature, gas mixture, and pH (suchas at about 20 to about 37° C., at about 6% to about 84% CO₂, and at apH between about 5 to about 9). In some embodiments, cells are grown at35° C. in an appropriate cell medium. In some embodiments, e.g.,cultures are cultured at approximately 28° C. in appropriate medium inshake cultures or fermentors until desired amount of isoprene productionis achieved. In some embodiments, the pH ranges for fermentation arebetween about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH8.0 or about 6.5 to about 7.0). Reactions may be performed underaerobic, anoxic, or anaerobic conditions based on the requirements ofthe host cells. Exemplary culture conditions for a given filamentousfungus are known in the art and may be found in the scientificliterature and/or from the source of the fungi such as the American TypeCulture Collection and Fungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used,such as the Fed-Batch system. Fed-Batch fermentation processes comprisea typical batch system with the exception that the carbon source isadded in increments as the fermentation progresses. Fed-Batch systemsare useful when catabolite repression is apt to inhibit the metabolismof the cells and where it is desirable to have limited amounts of carbonsource in the cell medium. Fed-batch fermentations may be performed withthe carbon source (e.g., glucose) in a limited or excess amount.Measurement of the actual carbon source concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen, and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used.Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or isoprene production. Forexample, one method maintains a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allows all other parametersto moderate. In other systems, a number of factors affecting growth canbe altered continuously while the cell concentration (e.g., theconcentration measured by media turbidity) is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, thecell loss due to media being drawn off is balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isopreneproduction.

In some embodiments, bottles of liquid culture are placed in shakers inorder to introduce oxygen to the liquid and maintain the uniformity ofthe culture. In some embodiments, an incubator is used to control thetemperature, humidity, shake speed, and/or other conditions in which aculture is grown. The simplest incubators are insulated boxes with anadjustable heater, typically going up to ˜65° C. More elaborateincubators can also include the ability to lower the temperature (viarefrigeration), or the ability to control humidity or CO₂ levels. Mostincubators include a timer; some can also be programmed to cycle throughdifferent temperatures, humidity levels, etc. Incubators can vary insize from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed toreplenish nutrients and/or avoid the build up of potentially harmfulmetabolic byproducts and dead cells. In the case of suspension cultures,cells can be separated from the media by centrifuging or filtering thesuspension culture and then resuspending the cells in fresh media. Inthe case of adherent cultures, the media can be removed directly byaspiration and replaced. In some embodiments, the cell medium allows atleast a portion of the cells to divide for at least or about 5, 10, 20,40, 50, 60, 65, or more cell divisions in a continuous culture (such asa continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trcpromoter) is used and a compound (such as IPTG) is not added to induceexpression of the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid(s) operably linked to the promoter. In some embodiments, a compound(such as IPTG) is added to induce expression of the isoprene synthase,DXS, IDI, or MVA pathway nucleic acid(s) operably linked to thepromoter.

Exemplary Methods for Decoupling Isoprene Production from Cell Growth

Desirably, carbon from the feedstock is converted to isoprene ratherthan to the growth and maintenance of the cells. In some embodiments,the cells are grown to a low to medium OD₆₀₀, then production ofisoprene is started or increased. This strategy permits a large portionof the carbon to be converted to isoprene.

In some embodiments, cells reach an optical density such that they nolonger divide or divide extremely slowly, but continue to make isoprenefor several hours (such as about 2, 4, 6, 8, 10, 15, 20, 25, 30, or morehours). For example, FIGS. 60A-67C illustrate that cells may continue toproduce a substantial amount of mevalonic acid or isoprene after thecells reach an optical density such that they no longer divide or divideextremely slowly. In some cases, the optical density at 550 nm decreasesover time (such as a decrease in the optical density after the cells areno longer in an exponential growth phase due to cell lysis), and thecells continue to produce a substantial amount of mevalonic acid orisoprene. In some embodiments, the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%) over a certain time period (such as greaterthan or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce isoprene at greater than or about 1, 10, 25, 50, 100, 150, 200,250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750,2,000, 2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram ofcells for the wet weight of the cells/hour (nmole/g_(wcm)/hr) duringthis time period. In some embodiments, the amount of isoprene is betweenabout 2 to about 5,000 nmole/g_(wcm)/hr, such as between about 2 toabout 100 nmole/g_(wcm)/hr, about 100 to about 500 nmole/g_(wcm)/hr,about 150 to about 500 nmole/g_(wcm)/hr, about 500 to about 1,000nmole/g_(wcm)/hr, about 1,000 to about 2,000 nmole/g_(wcm)/hr, or about2,000 to about 5,000 nmole/g_(wcm)/hr. In some embodiments, the amountof isoprene is between about 20 to about 5,000 nmole/g_(wcm)/hr, about100 to about 5,000 nmole/g_(wcm)/hr, about 200 to about 2,000nmole/g_(wcm)/hr, about 200 to about 1,000 nmole/g_(wcm)/hr, about 300to about 1,000 nmole/g_(wcm)/hr, or about 400 to about 1,000nmole/g_(wcm)/hr.

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsproduce a cumulative titer (total amount) of isoprene at greater than orabout 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800,900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000, 5,000,10,000, 50,000, 100,000, or more mg of isoprene/L of broth(mg/L_(broth), wherein the volume of broth includes the volume of thecells and the cell medium) during this time period. In some embodiments,the amount of isoprene is between about 2 to about 5,000 mg/L_(broth),such as between about 2 to about 100 mg/L_(broth), about 100 to about500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth), about 1,000 toabout 2,000 mg/L_(broth), or about 2,000 to about 5,000 mg/L_(broth). Insome embodiments, the amount of isoprene is between about 20 to about5,000 mg/L_(broth), about 100 to about 5,000 mg/L_(broth), about 200 toabout 2,000 mg/L_(broth), about 200 to about 1,000 mg/L_(broth), about300 to about 1,000 mg/L_(broth), or about 400 to about 1,000mg/L_(broth).

In some embodiments, the optical density at 550 nm of the cellsincreases by less than or about 50% (such as by less than or about 40,30, 20, 10, 5, or 0%) over a certain time period (such as greater thanor about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours), and the cellsconvert greater than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05,0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2,1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of thecarbon in the cell culture medium into isoprene during this time period.In some embodiments, the percent conversion of carbon into isoprene isbetween such as about 0.002 to about 4.0%, about 0.002 to about 3.0%,about 0.002 to about 2.0%, about 0.002 to about 1.6%, about 0.002 toabout 0.005%, about 0.005 to about 0.01%, about 0.01 to about 0.05%,about 0.05 to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%,about 0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about1.0%, or about 1.0 to about 1.6%. In some embodiments, the percentconversion of carbon into isoprene is between about 0.002 to about 0.4%,0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to about 0.3%,about 0.01 to about 0.3%, or about 0.05 to about 0.3%.

In some embodiments, isoprene is only produced in stationary phase. Insome embodiments, isoprene is produced in both the growth phase andstationary phase. In various embodiments, the amount of isopreneproduced (such as the total amount of isoprene produced or the amount ofisoprene produced per liter of broth per hour per OD₆₀₀) duringstationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40,50, or more times the amount of isoprene produced during the growthphase for the same length of time. In various embodiments, greater thanor about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99% or more of thetotal amount of isoprene that is produced (such as the production ofisoprene during a fermentation for a certain amount of time, such as 20hours) is produced while the cells are in stationary phase. In variousembodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 95, 99% or more of the total amount of isoprene that is produced(such as the production of isoprene during a fermentation for a certainamount of time, such as 20 hours) is produced while the cells divideslowly or not at all such that the optical density at 550 nm of thecells increases by less than or about 50% (such as by less than or about40, 30, 20, 10, 5, or 0%). In some embodiments, isoprene is onlyproduced in the growth phase.

In some embodiments, one or more MVA pathway, IDI, DXP, or isoprenesynthase nucleic acids are placed under the control of a promoter orfactor that is more active in stationary phase than in the growth phase.For example, one or more MVA pathway, IDI, DXP, or isoprene synthasenucleic acids may be placed under control of a stationary phase sigmafactor, such as RpoS. In some embodiments, one or more MVA pathway, IDI,DXP, or isoprene synthase nucleic acids are placed under control of apromoter inducible in stationary phase, such as a promoter inducible bya response regulator active in stationary phase.

Production of Isoprene within Safe Operating Ranges

The production of isoprene within safe operating levels according to itsflammability characteristics simplifies the design and construction ofcommercial facilities, vastly improves the ability to operate safely,and limits the potential for fires to occur. In particular, the optimalranges for the production of isoprene are within the safe zone, i.e.,the nonflammable range of isoprene concentrations. In one such aspect,the invention features a method for the production of isoprene withinthe nonflammable range of isoprene concentrations (outside theflammability envelope of isoprene).

Thus, computer modeling and experimental testing were used to determinethe flammability limits of isoprene (such as isoprene in the presence ofO₂, N₂, CO₂, or any combination of two or more of the foregoing gases)in order to ensure process safety. The flammability envelope ischaracterized by the lower flammability limit (LFL), the upperflammability limit (UFL), the limiting oxygen concentration (LOC), andthe limiting temperature. For a system to be flammable, a minimum amountof fuel (such as isoprene) must be in the presence of a minimum amountof oxidant, typically oxygen. The LFL is the minimum amount of isoprenethat must be present to sustain burning, while the UFL is the maximumamount of isoprene that can be present. Above this limit, the mixture isfuel rich and the fraction of oxygen is too low to have a flammablemixture. The LOC indicates the minimum fraction of oxygen that must alsobe present to have a flammable mixture. The limiting temperature isbased on the flash point of isoprene and is that lowest temperature atwhich combustion of isoprene can propagate. These limits are specific tothe concentration of isoprene, type and concentration of oxidant, inertspresent in the system, temperature, and pressure of the system.Compositions that fall within the limits of the flammability envelopepropagate combustion and require additional safety precautions in boththe design and operation of process equipment.

The following conditions were tested using computer simulation andmathematical analysis and experimental testing. If desired, otherconditions (such as other temperature, pressure, and permanent gascompositions) may be tested using the methods described herein todetermine the LFL, UFL, and LOC concentrations.

(1) Computer Simulation and Mathematical Analysis

Test Suite 1:

-   isoprene: 0 wt %-14 wt %-   O₂: 6 wt %-21 wt %-   N₂: 79 wt %-94 wt %    Test Suite 2:-   isoprene: 0 wt %-14 wt %-   O₂: 6 wt %-21 wt %-   N₂: 79 wt %-94 wt %-   Saturated with H₂O    Test Suite 3:-   isoprene: 0 wt %-14 wt %-   O₂: 6 wt %-21 wt %-   N₂: 79 wt %-94 wt %-   CO₂: 5 wt %-30 wt %    (2) Experimental Testing for Final Determination of Flammability    Limits    Test Suite 1:-   isoprene: 0 wt %-14 wt %-   O₂: 6 wt %-21 wt %-   N₂: 79 wt %-94 wt %    Test Suite 2:-   isoprene: 0 wt %-14 wt %-   O₂: 6 wt %-21 wt %-   N₂: 79 wt %-94 wt %-   Saturated with H₂O

Simulation software was used to give an estimate of the flammabilitycharacteristics of the system for several different testing conditions.CO₂ showed no significant affect on the system's flammability limits.Test suites 1 and 2 were confirmed by experimental testing. The modelingresults were in-line with the experimental test results. Only slightvariations were found with the addition of water.

The LOC was determined to be 9.5 vol % for an isoprene, O₂, N₂, and CO₂mixture at 40° C. and 1 atmosphere. The addition of up to 30% CO₂ didnot significantly affect the flammability characteristics of anisoprene, O₂, and N₂ mixture. Only slight variations in flammabilitycharacteristics were shown between a dry and water saturated isoprene,O₂, and N₂ system. The limiting temperature is about −54° C.Temperatures below about −54° C. are too low to propagate combustion ofisoprene.

In some embodiments, the LFL of isoprene ranges from about 1.5 vol. % toabout 2.0 vol %, and the UFL of isoprene ranges from about 2.0 vol. % toabout 12.0 vol. %, depending on the amount of oxygen in the system. Insome embodiments, the LOC is about 9.5 vol % oxygen. In someembodiments, the LFL of isoprene is between about 1.5 vol. % to about2.0 vol %, the UFL of isoprene is between about 2.0 vol. % to about 12.0vol. %, and the LOC is about 9.5 vol % oxygen when the temperature isbetween about 25° C. to about 55° C. (such as about 40° C.) and thepressure is between about 1 atmosphere and 3 atmospheres.

In some embodiments, isoprene is produced in the presence of less thanabout 9.5 vol % oxygen (that is, below the LOC required to have aflammable mixture of isoprene). In some embodiments in which isoprene isproduced in the presence of greater than or about 9.5 vol % oxygen, theisoprene concentration is below the LFL (such as below about 1.5 vol.%). For example, the amount of isoprene can be kept below the LFL bydiluting the isoprene composition with an inert gas (e.g., bycontinuously or periodically adding an inert gas such as nitrogen tokeep the isoprene composition below the LFL). In some embodiments inwhich isoprene is produced in the presence of greater than or about 9.5vol % oxygen, the isoprene concentration is above the UFL (such as aboveabout 12 vol. %). For example, the amount of isoprene can be kept abovethe UFL by using a system (such as any of the cell culture systemsdescribed herein) that produces isoprene at a concentration above theUFL. If desired, a relatively low level of oxygen can be used so thatthe UFL is also relatively low. In this case, a lower isopreneconcentration is needed to remain above the UFL.

In some embodiments in which isoprene is produced in the presence ofgreater than or about 9.5 vol % oxygen, the isoprene concentration iswithin the flammability envelope (such as between the LFL and the UFL).In some embodiments when the isoprene concentration may fall within theflammability envelope, one or more steps are performed to reduce theprobability of a fire or explosion. For example, one or more sources ofignition (such as any materials that may generate a spark) can beavoided. In some embodiments, one or more steps are performed to reducethe amount of time that the concentration of isoprene remains within theflammability envelope. In some embodiments, a sensor is used to detectwhen the concentration of isoprene is close to or within theflammability envelope. If desired, the concentration of isoprene can bemeasured at one or more time points during the culturing of cells, andthe cell culture conditions and/or the amount of inert gas can beadjusted using standard methods if the concentration of isoprene isclose to or within the flammability envelope. In particular embodiments,the cell culture conditions (such as fermentation conditions) areadjusted to either decrease the concentration of isoprene below the LFLor increase the concentration of isoprene above the UFL. In someembodiments, the amount of isoprene is kept below the LFL by dilutingthe isoprene composition with an inert gas (such as by continuously orperiodically adding an inert gas to keep the isoprene composition belowthe LFL).

In some embodiments, the amount of flammable volatiles other thanisoprene (such as one or more sugars) is at least about 2, 5, 10, 50,75, or 100-fold less than the amount of isoprene produced. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 100% (volume) oxygen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 100% (volume) oxygen. In someembodiments, the portion of the gas phase other than isoprene gascomprises between about 0% to about 99% (volume) nitrogen, such asbetween about 0% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, about 40% to about 50%, about 50% toabout 60%, about 60% to about 70%, about 70% to about 80%, about 90% toabout 90%, or about 90% to about 99% (volume) nitrogen.

In some embodiments, the portion of the gas phase other than isoprenegas comprises between about 1% to about 50% (volume) CO₂, such asbetween about 1% to about 10%, about 10% to about 20%, about 20% toabout 30%, about 30% to about 40%, or about 40% to about 50% (volume)CO₂.

In some embodiments, an isoprene composition also contains ethanol. Forexample, ethanol may be used for extractive distillation of isoprene,resulting in compositions (such as intermediate product streams) thatinclude both ethanol and isoprene. Desirably, the amount of ethanol isoutside the flammability envelope for ethanol. The LOC of ethanol isabout 8.7 vol %, and the LFL for ethanol is about 3.3 vol % at standardconditions, such as about 1 atmosphere and about 60° F. (NFPA 69Standard on Explosion Prevention Systems, 2008 edition, which is herebyincorporated by reference in its entirety, particularly with respect toLOC, LFL, and UFL values). In some embodiments, compositions thatinclude isoprene and ethanol are produced in the presence of less thanthe LOC required to have a flammable mixture of ethanol (such as lessthan about 8.7% vol %). In some embodiments in which compositions thatinclude isoprene and ethanol are produced in the presence of greaterthan or about the LOC required to have a flammable mixture of ethanol,the ethanol concentration is below the LFL (such as less than about 3.3vol. %).

In various embodiments, the amount of oxidant (such as oxygen) is belowthe LOC of any fuel in the system (such as isoprene or ethanol). Invarious embodiments, the amount of oxidant (such as oxygen) is less thanabout 60, 40, 30, 20, 10, or 5% of the LOC of isoprene or ethanol. Invarious embodiments, the amount of oxidant (such as oxygen) is less thanthe LOC of isoprene or ethanol by at least 2, 4, 5, or more absolutepercentage points (vol %). In particular embodiments, the amount ofoxygen is at least 2 absolute percentage points (vol %) less than theLOC of isoprene or ethanol (such as an oxygen concentration of less than7.5 vol % when the LOC of isoprene is 9.5 vol %). In variousembodiments, the amount of fuel (such as isoprene or ethanol) is lessthan or about 25, 20, 15, 10, or 5% of the LFL for that fuel.

Exemplary Production of Isoprene

In some embodiments, the cells are cultured in a culture medium underconditions permitting the production of isoprene by the cells. By “peakabsolute productivity” is meant the maximum absolute amount of isoprenein the off-gas during the culturing of cells for a particular period oftime (e.g., the culturing of cells during a particular fermentationrun). By “peak absolute productivity time point” is meant the time pointduring a fermentation run when the absolute amount of isoprene in theoff-gas is at a maximum during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the isoprene amount is measuredat the peak absolute productivity time point. In some embodiments, thepeak absolute productivity for the cells is about any of the isopreneamounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isopreneproduced per cell during the culturing of cells for a particular periodof time (e.g., the culturing of cells during a particular fermentationrun). By “peak specific productivity time point” is meant the time pointduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run) when the amountof isoprene produced per cell is at a maximum. The specific productivityis determined by dividing the total productivity by the amount of cells,as determined by optical density at 600 nm (OD600). In some embodiments,the isoprene amount is measured at the peak specific productivity timepoint. In some embodiments, the peak specific productivity for the cellsis about any of the isoprene amounts per cell disclosed herein.

By “cumulative total productivity” is meant the cumulative, total amountof isoprene produced during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the cumulative, total amount ofisoprene is measured. In some embodiments, the cumulative totalproductivity for the cells is about any of the isoprene amountsdisclosed herein.

By “relative detector response” refers to the ratio between the detectorresponse (such as the GC/MS area) for one compound (such as isoprene) tothe detector response (such as the GC/MS area) of one or more compounds(such as all C5 hydrocarbons). The detector response may be measured asdescribed herein, such as the GC/MS analysis performed with an Agilent6890 GC/MS system fitted with an Agilent HP-5MS GC/MS column (30 m×250μm; 0.25 μm film thickness). If desired, the relative detector responsecan be converted to a weight percentage using the response factors foreach of the compounds. This response factor is a measure of how muchsignal is generated for a given amount of a particular compound (thatis, how sensitive the detector is to a particular compound). Thisresponse factor can be used as a correction factor to convert therelative detector response to a weight percentage when the detector hasdifferent sensitivities to the compounds being compared. Alternatively,the weight percentage can be approximated by assuming that the responsefactors are the same for the compounds being compared. Thus, the weightpercentage can be assumed to be approximately the same as the relativedetector response.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, or more nmole of isoprene/gram of cells for the wet weight of thecells/hour (nmole/g_(wcm)/hr). In some embodiments, the amount ofisoprene is between about 2 to about 5,000 nmole/g_(wcm)/hr, such asbetween about 2 to about 100 nmole/g_(wcm)/hr, about 100 to about 500nmole/g_(wcm)/hr, about 150 to about 500 nmole/g_(wcm)/hr, about 500 toabout 1,000 nmole/g_(wcm)/hr, about 1,000 to about 2,000nmole/g_(wcm)/hr, or about 2,000 to about 5,000 nmole/g_(wcm)/hr. Insome embodiments, the amount of isoprene is between about 20 to about5,000 nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about200 to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, or about400 to about 1,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured asdisclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated byreference in its entirety, particularly with respect to the measurementof isoprene production. For example, two mL of headspace (e.g.,headspace from a culture such as 2 mL of culture cultured in sealedvials at 32° C. with shaking at 200 rpm for approximately 3 hours) areanalyzed for isoprene using a standard gas chromatography system, suchas a system operated isothermally (85° C.) with an n-octane/porasil Ccolumn (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2mercuric oxide reduction gas detector (Trace Analytical, Menlo Park,Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A:2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to the measurement of isoprene production).The gas chromatography area units are converted to nmol isoprene via astandard isoprene concentration calibration curve. In some embodiments,the value for the grams of cells for the wet weight of the cells iscalculated by obtaining the A₆₀₀ value for a sample of the cell culture,and then converting the A₆₀₀ value to grams of cells based on acalibration curve of wet weights for cell cultures with a known A₆₀₀value. In some embodiments, the grams of the cells is estimated byassuming that one liter of broth (including cell medium and cells) withan A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is alsodivided by the number of hours the culture has been incubating for, suchas three hours.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wetweight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amountof isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such asbetween about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 toabout 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. Insome embodiments, the amount of isoprene is between about 20 to about5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 toabout 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculatedby multiplying the value for isoprene production in the units ofnmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5below).

In some embodiments, the cells in culture produce a cumulative titer(total amount) of isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, ormore mg of isoprene/L of broth (mg/L_(broth), wherein the volume ofbroth includes the volume of the cells and the cell medium). In someembodiments, the amount of isoprene is between about 2 to about 5,000mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth),about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000mg/L_(broth). In some embodiments, the amount of isoprene is betweenabout 20 to about 5,000 mg/L_(broth), about 100 to about 5,000mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspacefrom shake flask or similar cultures can be measured by taking a 1 mlsample from the cell culture at an OD₆₀₀ value of approximately 1.0,putting it in a 20 mL vial, incubating for 30 minutes, and thenmeasuring the amount of isoprene in the headspace (as described, forexample, in Example 10, part II). If the OD₆₀₀ value is not 1.0, thenthe measurement can be normalized to an OD₆₀₀ value of 1.0 by dividingby the OD₆₀₀ value. The value of mg isoprene/L headspace can beconverted to mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by afactor of 38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can bemultiplied by the number of hours and the OD₆₀₀ value to obtain thecumulative titer in units of mg of isoprene/L of broth.

The instantaneous isoprene production rate in mg/L_(broth)/hr in afermentor can be measured by taking a sample of the fermentor off-gas,analyzing it for the amount of isoprene (in units such as mg of isopreneper L_(gas)) as described, for example, in Example 10, part II andmultiplying this value by the rate at which off-gas is passed thougheach liter of broth (e.g., at 1 vvm (volume of air/volume ofbroth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1mg/L_(gas) corresponds to an instantaneous production rate of 60mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in the unitsmg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specificrate in units of mg/L_(broth)/hr/OD. The average value of mgisoprene/L_(gas) can be converted to the total product productivity(grams of isoprene per liter of fermentation broth, mg/L_(broth)) bymultiplying this average off-gas isoprene concentration by the totalamount of off-gas sparged per liter of fermentation broth during thefermentation. Thus, an average off-gas isoprene concentration of 0.5mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total productconcentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0,3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culturemedium into isoprene. In some embodiments, the percent conversion ofcarbon into isoprene is between such as about 0.002 to about 4.0%, about0.002 to about 3.0%, about 0.002 to about 2.0%, about 0.002 to about1.6%, about 0.002 to about 0.005%, about 0.005 to about 0.01%, about0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%,about 0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In someembodiments, the percent conversion of carbon into isoprene is betweenabout 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%,about 0.005 to about 0.3%, about 0.01 to about 0.3%, or about 0.05 toabout 0.3%.

The percent conversion of carbon into isoprene (also referred to as “%carbon yield”) can be measured by dividing the moles carbon in theisoprene produced by the moles carbon in the carbon source (such as themoles of carbon in batched and fed glucose and yeast extract). Thisnumber is multiplied by 100% to give a percentage value (as indicated inEquation 1).% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon incarbon source)*100  Equation 1

For this calculation, yeast extract can be assumed to contain 50% w/wcarbon. As an example, for the 500 liter described in Example 16, partVIII, the percent conversion of carbon into isoprene can be calculatedas shown in Equation 2.Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 gglucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12mol/g)]*100=0.042%  Equation 2

For the two 500 liter fermentations described herein (Example 16, partsVII and VIII), the percent conversion of carbon into isoprene wasbetween 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14liter systems as described herein. Example 19, part V describes the1.53% conversion of carbon to isoprene using the methods describedherein.

One skilled in the art can readily convert the rates of isopreneproduction or amount of isoprene produced into any other units.Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene Production (Total and Specific)1 g isoprene/L _(broth)/hr=14.7 mmol isoprene/L_(broth)/hr(totalvolumetric rate)  Equation 31 nmol isoprene/g_(wcm)/hr=1 nmol isoprene/L_(broth)/hr/OD₆₀₀(Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa wet cell weight of 1 gram.)  Equation 41 nmol isoprene/g_(wcm)/hr=68.1 ng isoprene/g_(wcm)/hr(given themolecular weight of isoprene)  Equation 51 nmol isoprene/L_(gas) O₂/hr=90 nmol isoprene/L_(broth)/hr(at an O₂flow rate of 90 L/hr per L of culture broth)  Equation 61 ug isoprene/L_(gas) isoprene in off-gas=60 ug isoprene/L_(broth)/hr ata flow rate of 60 L_(gas) per L_(broth)(1 vvm)  Equation 7Units for Titer (Total and Specific)1 nmol isoprene/mg cell protein=150 nmol isoprene/L_(broth)/OD₆₀₀(Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa total cell protein of approximately 150 mg) (specificproductivity)  Equation 81 g isoprene/L_(broth)=14.7 mmol isoprene/L_(broth)(totaltiter)  Equation 9

If desired, Equation 10 can be used to convert any of the units thatinclude the wet weight of the cells into the corresponding units thatinclude the dry weight of the cells.Dry weight of cells=(wet weight of cells)/3.3  Equation 10

If desired, Equation 11 can be used to convert between units of ppm andug/L. In particular, “ppm” means parts per million defined in terms ofug/g (w/w). Concentrations of gases can also be expressed on avolumetric basis using “ppmv” (parts per million by volume), defined interms of uL/L (vol/vol). Conversion of ug/L to ppm (e.g., ug of analyteper g of gas) can be performed by determining the mass per L of off-gas(i.e., the density of the gas). For example, a liter of air at standardtemperature and pressure (STP; 101.3 kPa (1 bar) and 273.15K) has adensity of approximately 1.29 g/L. Thus, a concentration of 1 ppm (ug/g)equals 1.29 ug/L at STP (equation 11). The conversion of ppm (ug/g) toug/L is a function of both pressure, temperature, and overallcomposition of the off-gas.1 ppm(ug/g)equals 0.83 ug/L at standard temperature and pressure(STP;101.3 kPa (1 bar) and 273.15K).  Equation 11

Conversion of ug/L to ppmv (e.g., uL of analyte per L of gas) can beperformed using the Universal Gas Law (equation 12). For example, anoff-gas concentration of 1000 ug/L_(gas) corresponds to 14.7umol/L_(gas). The universal gas constant is 0.082057 L.atm K⁻¹ mol⁻¹, sousing equation 12, the volume occupied by 14.7 umol of HG at STP isequal to 0.329 mL. Therefore, the concentration of 1000 ug/L HG is equalto 329 ppmv or 0.0329% (v/v) at STP.PV=nRT, where “P” is pressure, “V” is volume, “n” is moles of gas, “R”is the Universal gas constant, and “T” is temperature inKelvin.  Equation 12

The amount of impurities in isoprene compositions are typically measuredherein on a weight per volume (w/v) basis in units such as ug/L. Ifdesired, measurements in units of ug/L can be converted to units ofmg/m³ using equation 13.1 ug/L=1 mg/m³  Equation 13

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduces an amount of isoprene that is at least or about 2-fold, 3-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold,400-fold, or greater than the amount of isoprene produced from acorresponding cell grown under essentially the same conditions withoutthe heterologous nucleic acid encoding the isoprene synthasepolypeptide.

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptide andone or more heterologous nucleic acids encoding a DXS, IDI, and/or MVApathway polypeptide produces an amount of isoprene that is at least orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of isopreneproduced from a corresponding cell grown under essentially the sameconditions without the heterologous nucleic acids.

In some embodiments, the isoprene composition comprises greater than orabout 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.In some embodiments, the composition has a relative detector response ofgreater than or about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96,99.97, 99.98, 99.99, or 100% for isoprene compared to the detectorresponse for all C5 hydrocarbons in the composition. In someembodiments, the isoprene composition comprises between about 99.90 toabout 99.92, about 99.92 to about 99.94, about 99.94 to about 99.96,about 99.96 to about 99.98, about 99.98 to 100% isoprene by weightcompared to the total weight of all C5 hydrocarbons in the composition.

In some embodiments, the isoprene composition comprises less than orabout 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than isoprene (such1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) by weight compared tothe total weight of all C5 hydrocarbons in the composition. In someembodiments, the composition has a relative detector response of lessthan or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,0.0005, 0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other thanisoprene compared to the detector response for all C5 hydrocarbons inthe composition. In some embodiments, the composition has a relativedetector response of less than or about 0.12, 0.10, 0.08, 0.06, 0.04,0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne,trans-piperylene, cis-piperylene, pent-4-ene-1-yne,trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne compared to the detectorresponse for all C5 hydrocarbons in the composition. In someembodiments, the isoprene composition comprises between about 0.02 toabout 0.04%, about 0.04 to about 0.06%, about 0.06 to 0.08%, about 0.08to 0.10%, or about 0.10 to about 0.12% C5 hydrocarbons other thanisoprene (such 1,3-cyclopentadiene, cis-1,3-pentadiene,trans-1,3-pentadiene, 1-pentyne, 2-pentyne, 1-pentene,2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene, cis-piperylene,pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne) byweight compared to the total weight of all C5 hydrocarbons in thecomposition.

In some embodiments, the isoprene composition comprises less than orabout 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of acompound that inhibits the polymerization of isoprene for any compoundin the composition that inhibits the polymerization of isoprene. In someembodiments, the isoprene composition comprises between about 0.005 toabout 50, such as about 0.01 to about 10, about 0.01 to about 5, about0.01 to about 1, about 0.01 to about 0.5, or about 0.01 to about 0.005ug/L of a compound that inhibits the polymerization of isoprene for anycompound in the composition that inhibits the polymerization ofisoprene. In some embodiments, the isoprene composition comprises lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005ug/L of a hydrocarbon other than isoprene (such 1,3-cyclopentadiene,cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne,1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene,cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, orcis-pent-3-ene-1-yne). In some embodiments, the isoprene compositioncomprises between about 0.005 to about 50, such as about 0.01 to about10, about 0.01 to about 5, about 0.01 to about 1, about 0.01 to about0.5, or about 0.01 to about 0.005 ug/L of a hydrocarbon other thanisoprene. In some embodiments, the isoprene composition comprises lessthan or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005ug/L of a protein or fatty acid (such as a protein or fatty acid that isnaturally associated with natural rubber).

In some embodiments, the isoprene composition comprises less than orabout 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of alphaacetylenes, piperylenes, acetonitrile, or 1,3-cyclopentadiene. In someembodiments, the isoprene composition comprises less than or about 5, 1,0.5, 0.1, 0.05, 0.01, or 0.005 ppm of sulfur or allenes. In someembodiments, the isoprene composition comprises less than or about 30,20, 15, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes(such as pentyne-1, butyne-2, 2MB1-3yne, and 1-pentyne-4yne). In someembodiments, the isoprene composition comprises less than or about 2000,1000, 500, 200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or0.005 ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g.,cyclic C10 compounds derived from the dimerization of two isopreneunits).

In some embodiments, the composition comprises greater than about 2 mgof isoprene, such as greater than or about 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg ofisoprene. In some embodiments, the composition comprises greater than orabout 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene. Insome embodiments, the amount of isoprene in the composition is betweenabout 2 to about 5,000 mg, such as between about 2 to about 100 mg,about 100 to about 500 mg, about 500 to about 1,000 mg, about 1,000 toabout 2,000 mg, or about 2,000 to about 5,000 mg. In some embodiments,the amount of isoprene in the composition is between about 20 to about5,000 mg, about 100 to about 5,000 mg, about 200 to about 2,000 mg,about 200 to about 1,000 mg, about 300 to about 1,000 mg, or about 400to about 1,000 mg. In some embodiments, greater than or about 20, 25,30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile organicfraction of the composition is isoprene.

In some embodiments, the composition includes ethanol. In someembodiments, the composition includes between about 75 to about 90% byweight of ethanol, such as between about 75 to about 80%, about 80 toabout 85%, or about 85 to about 90% by weight of ethanol. In someembodiments in which the composition includes ethanol, the compositionalso includes between about 4 to about 15% by weight of isoprene, suchas between about 4 to about 8%, about 8 to about 12%, or about 12 toabout 15% by weight of isoprene.

Exemplary Isoprene Purification Methods

In some embodiments, any of the methods described herein further includerecovering the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be recovered usingstandard techniques, such as gas stripping, membrane enhancedseparation, fractionation, adsorption/desorption, pervaporation, thermalor vacuum desorption of isoprene from a solid phase, or extraction ofisoprene immobilized or absorbed to a solid phase with a solvent (see,for example, U.S. Pat. Nos. 4,703,007 and 4,570,029, which are eachhereby incorporated by reference in their entireties, particularly withrespect to isoprene recovery and purification methods). In particular,embodiments, extractive distillation with an alcohol (such as ethanol,methanol, propanol, or a combination thereof) is used to recover theisoprene. In some embodiments, the recovery of isoprene involves theisolation of isoprene in a liquid form (such as a neat solution ofisoprene or a solution of isoprene in a solvent). Gas stripping involvesthe removal of isoprene vapor from the fermentation off-gas stream in acontinuous manner. Such removal can be achieved in several differentways including, but not limited to, adsorption to a solid phase,partition into a liquid phase, or direct condensation (such ascondensation due to exposure to a condensation coil or do to an increasein pressure). In some embodiments, membrane enrichment of a diluteisoprene vapor stream above the dew point of the vapor resulting in thecondensation of liquid isoprene. In some embodiments, the isoprene iscompressed and condensed.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from the fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent.

In some embodiments, any of the methods described herein further includepurifying the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, at least a portion of the gas phase remaining afterone or more recovery steps for the removal of isoprene is recycled byintroducing the gas phase into a cell culture system (such as afermentor) for the production of isoprene.

In some embodiments, any of the methods described herein further includepolymerizing the isoprene. For example, standard methods can be used topolymerize the purified isoprene to form cis-polyisoprene or other downstream products using standard methods. Accordingly, the invention alsofeatures a tire comprising polyisoprene, such as cis-1,4-polyisopreneand/or trans-1,4-polyisoprene made from any of the isoprene compositionsdisclosed herein.

The following Examples are provided to illustrate but not limit theinvention.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. Unless indicated otherwise, temperature is in degreesCentigrade and pressure is at or near atmospheric. The foregoingexamples and detailed description are offered by way of illustration andnot by way of limitation. All publications, patent applications, andpatents cited in this specification are herein incorporated by referenceas if each individual publication, patent application, or patent werespecifically and individually indicated to be incorporated by reference.In particular, all publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies which might be used in connection withthe invention. Although the foregoing invention has been described insome detail by way of illustration and example for purposes of clarityof understanding, it will be readily apparent to those of ordinary skillin the art in light of the teachings of this invention that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims.

Example 1 Expression Constructs and Strains

I. Construction of Plasmids Encoding Mevalonate Kinase.

A construct encoding the Methanosarcina mazei lower MVA pathway(Accession numbers NC_(—)003901.1, NC_(—)003901.1, NC_(—)003901.1, andNC_(—)003901.1, which are each hereby incorporated by reference in theirentireties) was synthesized with codon optimization for expression in E.coli. This construct is named M. mazei archaeal Lower Pathway operon(FIGS. 46A-46C) and encodes M. mazei MVK, a putative decarboxylase, IPK,and IDI enzymes. The gene encoding MVK (Accession number NC_(—)003901.1)was PCR amplified using primers MCM165 and MCM177 (Table 4) using theStrategene Herculase II Fusion kit according to the manufacturer'sprotocol using 30 cycles with an annealing temperature of 55° C. andextension time of 60 seconds. This amplicon was purified using a QiagenPCR column and then digested at 37° C. in a 10 uL reaction with PmeI (inthe presence of NEB buffer 4 and BSA). After one hour, NsiI and Rochebuffer H were added for an additional hour at 37° C. The digested DNAwas purified over a Qiagen PCR column and ligated to a similarlydigested and purified plasmid MCM29 in an 11 uL reaction 5 uL RocheQuick Ligase buffer 1, 1 uL buffer 2, 1 uL plasmid, 3 uL amplicon, and 1uL ligase (1 hour at room temperature). MCM29 is pTrcKudzukan. Theligation reaction was introduced into Invitrogen TOP10 cells andtransformants selected on LA/kan50 plates incubated at 37° C. overnight.The MVK insert in the resulting plasmid MCM382 was sequenced (FIGS.47A-47C).

Using the method described above for plasmid MCM382,pTrcKudzu-MVK(mazei), additional plasmids were constructed with MVKgenes from different source organisms (Table 5 and FIGS. 59A-59C).

TABLE 5 Plasmid encoding MVK from Saccharomyces cerevisiae. SourceForward Reverse Final Organism PCR Template Primer Primer PlasmidSaccharomyces pTrcKK MCM170 MCM171 MCM383 cerevisiae (described herein)II. Creation of Strains Overexpressing Mevalonate Kinase and IsopreneSynthase.

Plasmid MCM382 was transformed into MCM331 cells (which containchromosomal construct gi1.2KKDyI encoding S. cerevisiae mevalonatekinase, mevalonate phosphate kinase, mevalonate pyrophosphatedecarboxylase, and IPP isomerase) that had been grown to midlog in LBmedium and washed three times in iced, sterile water. 1 uL of DNA wasadded to 50 uL of cell suspension, and this mixture was electroporatedin a 2 mm cuvette at 2.5 volts, 25 uFd followed immediately by recoveryin 500 uL LB medium for one hour at 37° C. Transformant was selected onLA/kan50 and named MCM391. Plasmid MCM82 was introduced into this strainby the same electroporation protocol followed by selection onLA/kan50/spec50. The resulting strain MCM401 contains a cmp-markedchromosomal construct gi1.2KKDyI, kan-marked plasmid MCM382, andspec-marked plasmid MCM82 (which is pCL PtrcUpperPathway encoding E.faecalis mvaE and mvaS).

Production strains analogous to MCM401 were generated for each of thefour plasmids detailed in Table 5 using the methods described above forMCM401. MCM331 was transformed with plasmid MCM379, 380, 381, or 383,and then selected on LA+kan50. The resulting strains were transformedwith MCM82 and selected on LA+kan50+spec50.

TABLE 6 Strains overexpressing mevalonate kinase and isoprene synthaseStrain MCM331 Plasmid Strain MCM331 transformed with pTrcKudzu-transformed with pTrcKudzuMVK then MVK Source MVK pTrcKudzuMVKtransformed with MCM82 Methanosarcina MCM382 MCM391 MCM401 mazeiSaccharomyces MCM383 MCM392 MCM402 cerevisiae Strain MCM333 PlasmidStrain MCM333 transformed with pTrcKudzu- transformed with pTrcKudzuMVKthen MVK Source MVK pTrcKudzuMVK transformed with MCM82 MethanosarcinaMCM382 MCM396 MCM406 mazei Saccharomyces MCM383 MCM397 MCM407 cerevisiae

Additional strain information is provided below.

-   MCM382: E. coli BL21 (lambdaDE3) pTrcKudzuMVK(M. mazei)GI1.2KKDyI-   MCM391: MCM331 pTrcKudzuMVK(M. mazei)-   MCM401: MCM331pTrcKudzuMVK(M. mazei)pCLPtrcUpperpathway-   MCM396: MCM333pTrcKudzuMVK(M. mazei)-   MCM406: MCM333pTrcKudzuMVK(M. mazei)pCLPtrcUpperpathway    III. Construction of Plasmid MCM376-MVK from M. mazei Archaeal Lower    in pET200D.

The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS.46A-46C) was PCR amplified using primers MCM161 and MCM162 (Table 4)using the Invitrogen Platinum HiFi PCR mix. 45 uL of PCR mix wascombined with 1 uL template, 1 uL of each primer at 10 uM, and 2 uLwater. The reaction was cycled as follows: 94° C. for 2:00 minutes; 30cycles of 94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C.for 1:15 minutes; and then 72° C. for 7:00 minutes, and 4° C. untilcool. 3 uL of this PCR reaction was ligated to Invitrogen pET200Dplasmid according to the manufacturer's protocol. 3 uL of this ligationwas introduced into Invitrogen TOP10 cells, and transformants wereselected on LA/kan50. A plasmid from a transformant was isolated and theinsert sequenced, resulting in MCM376 (FIGS. 57A-57C).

VI. Construction of pDu5 Expressing S. cerevisiae MVK

The S. cerevisiae MVK was cloned into pET16b from Invitrogen as follows(Table 7). The MVK enzyme from S. cerevisiae was PCR amplified withHg-MVK-F2-NdeI and Hg-MVK-R2-NdeI primers using Stratagene Pfu UltraIIFusion DNA Polymerase Kit according to manufacturer's protocol, andpMVK1 (described herein) as the template DNA. The following cycleparameter was used for the reaction (95° C. for 2 minutes, 29 cycles(95° C. for 20 seconds, 55° C. for 20 seconds, 72° C. for 21 sececonds),72° C. for 3 minutes, and 4° C. until cool) using an MastercyclerGradient Machine).

As a result, a 1.352 kb MVK PCR fragment was obtained and was gelpurified using Qiagen's gel purification kit. The purified PCR productwas digested with NdeI restriction enzyme. The digested DNA was purifiedover Qiagen PCR column. 5 uL of purified PCR product was ligated to 1 uLof pET-16b vector that was previously digested with NdeI and thentreated with SAP (Shrimp Alkaline Phosphatase). A New England BioLab(NEB) T4 ligase kit was used for ligation at approximately 16° C.overnight according to manufacturer's protocol.

5 uL of overnight ligation mixture was transformed into Invitrogen TOP10cells. The transformation was carried on ice for a 30 minute incubationfollowed by a 30 second heat shock at approximately 42° C. and a 1 hourrecovery in 1 ml LB at approximately 37° C. The transformation wasselected on LA/Carb50 incubated at approximately 37° C. overnight.Plasmids from transformants were isolated and the insert sequenced withT7 promoter and T7 terminator using Quintara Bio Sequencing Service. Theresulting plasmid for S. cerevisiae MVK in pET-16b vector is called pDu5(FIGS. 126A and 126B).

Once the sequence is verified, 1 ul of plasmid (pDu5) is thentransformed into BL21 pLysS host strain. Transformants are selected onLA/Carb50 plates and incubated at approximately 37° C. The resultingexpression strain is called MD08-MVK.

TABLE 7 Plasmid and Strain overexpressing mevalonate kinase For. Rev.Expression Template Primer Primer Plasmid Strain S. cerevisiae pMVK1Hg-MVK- Hg-MVK- pDu5 MD08- F2-NdeI R2-NdeI MVKV. Creation of Expression Strain MCM378.

Plasmid MCM376 was transformed into Invitrogen OneShot BL21 Star (DE3)cells according to the manufacturer's protocol. Transformant MCM378 wasselected on LA/kan50. Additional strains were created using the sameprotocol and are listed in the Table 7. Invitrogen OneShot BL21(DE3)pLysS transformed with the indicated plasmid and selected on LA andcarb50 cmp35 for MD08-MVK were used.

VI. Construction of Plasmid pCLPtrcUpperPathwayHGS2

The gene encoding isoprene synthase from Pueraria lobata wasPCR-amplified using primers NsiI-RBS-HGS F(cttgATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID NO:113) and pTrcR(CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:114), and pTrcKKDyIkIS (MCM118) as atemplate. The resulting PCR product was restriction-digested with NsiIand PstI and gel-purified using the Qiagen QIAquick Gel Extraction kitusing standard methods. MCM82 (pCL PtrcUpperPathway) wasrestriction-digested with PstI and dephosphorylated using rAPid alkalinephosphatase (Roche). These DNA pieces were ligated together using T4ligase and the ligation reaction was transformed in E. coli Top10electrocompetent cells (Invitrogen). Plasmid was prepared from sixclones using the Qiagen QiaPrep Spin MiniPrep kit. The plasmids weredigested with restriction enzymes EcoRV and MluI, and a clone in whichthe insert had the right orientation (i.e., gene oriented in the sameway as the pTrc promoter) was identified. The resulting plasmidpCLPtrcUpperPathwayHGS2 (FIGS. 112A-112D) was found to produce isoprenein E. coli Top10, using a headspace assay described herein, thusvalidating the functionality of the expression construct.

TABLE 4 Oligonucleotides Hg-MVK-F2-NdeI cagcagcagCATATGtcattaccgttcttaacttc (SEQ ID NO: 115) Hg-MVK-R2-NdeI cagcagcagCATATGgcctatcgcaaattagcttatg (SEQ ID NO: 116) MCM161 M. mazei MVK  CACCATGGTATCCTGTTCTGCGfor (SEQ ID NO: 117) MCM162 M. mazei MVK  TTAATCTACTTTCAGACCTTGC rev(SEQ ID NO: 118) MCM165 M. mazei MVK  gcgaacgATGCATaaaggaggtaaaaaafor w/RBS acATGGTATCCTGTTCTGCGCCGGGTAA GATTTACCTG (SEQ ID NO: 119)MCM170 S. cerevisiae  gcgaacgATGCATaaaggaggtaaaaaa MVK for w/RBSacATGTCATTACCGTTCTTAACTTCTGC A (SEQ ID NO: 120) MCM171 S. cerevisiae gggcccgtttaaactttaactagactCT MVK rev w/RBS GCAGTTATGAAGTCCATGGTAAATTCGTGT (SEQ ID NO: 121) MCM177 M. mazei MVK  gggcccgtttaaactttaactagactTTrev Pst AATCTACTTTCAGACCTTGC (SEQ ID NO: 122)

Example 2 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 20 mL Batch Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 13.6 g, KH₂PO₄ 13.6g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g, and 1000× Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with ammonium hydroxide (30%) andbrought to volume. Media was filter sterilized with a 0.22 micronfilter. Glucose 2.5 g and antibiotics were added after sterilization andpH adjustment.

1000× Trace Metal Solution:

1000× Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component wasdissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then broughtto volume and filter sterilized with a 0.22 micron filter.

Strains:

MCM343 cells are BL21 (DE3) E. coli cells containing the upper mevalonicacid (MVA) pathway (pCL Upper), the integrated lower MVA pathway(gi1.2KKDyI), and isoprene synthase from Kudzu (pTrcKudzu). The S.cerevisiae MVK gene is present only as one copy on the chromosome of theMCM343 cells and is controlled by a weak promoter. The expression levelof isoprene synthase may not be limiting in the MCM343 cells. Theisoprene synthase gene has the same plasmid backbone and promoter as inthe MCM401 cells.

MCM401 cells are BL21 (DE3) E. coli cells containing the upper mevalonicacid (MVA) pathway (pCL Upper), the integrated lower MVA pathway(gi1.2KKDyI), and high expression of mevalonate kinase from M. mazei andisoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). The M. mazei MVKgene is present in multiple copies on a plasmid in the MCM401 cells(˜30-50 copies/cell) and is under a stronger promoter than the S.cerevisiae MVK gene. Based on this information, the MVK protein level inthe MCM401 cells is expected to be at least about 30 to 50 fold higherthan the level in the MCM343 cells. The expression level of isoprenesynthase may not be limiting in the MCM401 cells. The isoprene synthasegene shares the same plasmid backbone and promoter as the MCM343 cells.In addition, the amount of isoprene synthase made is higher in theMCM401 cells, and the protein level of the isoprene synthase was notdependent upon the inhibition of MVK.

Isoprene production was analyzed by growing the strains in 100 mLbioreactors with a 20 mL working volume at a temperature of 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 30° C. A singlecolony was inoculated into media and grown overnight. The bacteria werediluted into 20 mL of media to reach an optical density of 0.05 measuredat 550 nm. The 100 mL bioreactors were sealed, and air was pumpedthrough at a rate of 8 mL/min. Adequate agitation of the media wasobtained by stirring at 600 rpm using magnetic stir bars. The off-gasfrom the bioreactors was analyzed using an on-line Hiden HPR-20 massspectrometer. Masses corresponding to isoprene, CO₂, and other gassesnaturally occurring in air were monitored. Accumulated isoprene and CO₂production were calculated by summing the concentration (in percent) ofthe respective gasses over time. Atmospheric CO₂ was subtracted from thetotal in order to estimate the CO₂ released due to metabolic activity.

Isoprene production from a strain expressing the full mevalonic acidpathway and Kudzu isoprene synthase (MCM343) was compared to a strainthat in addition over-expressed MVK from M. mazei and Kudzu isoprenesynthase (MCM401) in 100 mL bioreactors. The bacteria were grown underidentical conditions in defined media with glucose as carbon source.Induction of isoprene production was achieved by addingisopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a final concentrationof either 100 uM or 200 uM. Off-gas measurements revealed that thestrain over-expressing both MVK and isoprene synthase (MCM401) producedsignificantly more isoprene compared to the strain expressing only themevalonic acid pathway and Kudzu isoprene synthase (MCM343) as shown inFIGS. 113A-113D. At 100 uM induction, the MCM401 strain produced 2-foldmore isoprene compared to the MCM343 strain. At 200 uM IPTG induction,the MCM401 strain produced 3.4-fold more isoprene when compared to theMCM343 strain. Analysis of CO₂ in the off-gas from the bioreactors,which is a measure of metabolic activity, indicates that metabolicactivity was independent from IPTG induction and isoprene production.

Example 3 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to innoculate 5-L of cellmedium in the 15-L bioreactor. In particular, the 15-L bioreactor had aninitial working volume of 5 L. The liquid volume increases throughoutthe fermentation (such as to approximately 10 liters).

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 68 hour fermentation was 3.8 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 51 uM when the optical density at 550nm (OD₅₅₀) reached a value of 9. The IPTG concentration was raised to 88uM when OD₅₅₀ reached 149. Additional IPTG additions raised theconcentration to 119 uM at OD₅₅₀=195 and 152 uM at OD₅₅₀=210. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 114. Theisoprene level in the off gas from the bioreactor was determined using aHiden mass spectrometer. The isoprene titer increased over the course ofthe fermentation to a final value of 23.8 g/L (FIG. 115). The totalamount of isoprene produced during the 68 hour fermentation was 227.2 gand the time course of production is shown in FIG. 116. The molar yieldof utilized carbon that went into producing isoprene during fermentationwas 13.0%. The weight percent yield of isoprene from glucose was 6.3%.

Example 4 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuCO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to innoculate 5-L of cellmedium in the 15-L bioreactor. The liquid volume increases throughoutthe fermentation (such as to approximately 10 liters).

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 55 hour fermentation was 1.9 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 111 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 193 uM when OD₅₅₀ reached 155. TheOD₅₅₀ profile within the bioreactor over time is shown in FIG. 130. Theisoprene level in the off gas from the bioreactor was determined using aHiden mass spectrometer. The isoprene titer increased over the course ofthe fermentation to a final value of 19.5 g/L (FIG. 131). The totalamount of isoprene produced during the 55 hour fermentation was 133.8 g,and the time course of production is shown in FIG. 132. Instantaneousvolumetric productivity levels reached values as high as 1.5 gisoprene/L broth/hr (FIG. 133). Instantaneous yield levels reached ashigh as 17.7% w/w (FIG. 134). The molar yield of utilized carbon thatwent into producing isoprene during fermentation was 15.8%. The weightpercent yield of isoprene from glucose over the entire fermentation was7.4%.

Example 5 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from M. mazei, and Isoprene Synthasefrom Kudzu and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuCO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from M. mazeiand isoprene synthase from Kudzu (pTrcKudzuMVK(M. mazei)). Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to innoculate 5-L of cellmedium in the 15-L bioreactor. The liquid volume increases throughoutthe fermentation (such as to approximately 10 liters).

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 55 hour fermentation was 2.2 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 51 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. Inaddition to the IPTG spike, at OD₅₅₀=10 a constant feed began anddelivered 164 mg of IPTG over 18 hours. The OD₅₅₀ profile within thebioreactor over time is shown in FIG. 135. The isoprene level in the offgas from the bioreactor was determined using a Hiden mass spectrometer.The isoprene titer increased over the course of the fermentation to afinal value of 22.0 g/L (FIG. 117). The total amount of isopreneproduced during the 55 hour fermentation was 170.5 g and the time courseof production is shown in FIG. 118. The molar yield of utilized carbonthat went into producing isoprene during fermentation was 16.6%. Theweight percent yield of isoprene from glucose over the entirefermentation was 7.7%.

Example 6 Over-Expression of Mevalonate Kinase and Isoprene Synthase inE. coli Harboring the MVA Pathway

Over-expression of both mevalonate kinase and isoprene synthase resultsin high specific productivity of isoprene production by E. coliharboring the MVA pathway.

I. Construction of Plasmid MCM94

Plasmid pTrcHis2B (Invitrogen) was digested for 2 hours at 30° C. in 10uL containing ApaI (Roche) and Roche BufferA. The reaction was broughtto a total of 30 uL containing 1× Roche Buffer H and 2 uL PstI (Roche)and incubated for 1 hour at 37° C. The 996 bp fragment containing thepTrc promoter region was gel purified from an Invitrogen E-gel (1.2%)using a Qiagen Gel Purification spin column according to themanufacturer's protocol.

Plasmid MCM29 was digested as described above, and the 3338 bp fragmentcontaining the origin and kanR genes was gel purified as describedabove. The two fragments (3 uL pTrcHis2B fragment, 1 uL MCM29 fragment)were ligated for 1 hour at room temperature in a 20 uL reactionfollowing the Roche Rapid DNA Ligation kit protocol. 5 uL of thisligation reaction was used to transform Invitrogen TOP10 chemicallycompetent cells according to the manufacturer's protocol. Transformantswere selected on LA and kanamycin50 ppm. Plasmids were isolated byQiagen Spin Miniprep from several colonies which had been grownovernight in 5 mL LB and kan50. A clone with the pTrc promoter but nokudzu isoprene synthase gene was frozen as MCM94 (FIGS. 119A-119C).

II. Construction of Strains MCM433, 437, and 438

Plasmid pCL PtrcUpperHGS2 (Construction of this plasmid is described inExample 1 part VI) was transformed into MCM331 by electroporation asdescribed herein for expression strain MCM401. Transformant MCM433 wasselected on LA and spectinomycin 50 ppm. Strain MCM433 was subsequentlytransformed with either plasmid MCM94 (described above) or MCM376 andselected on LA, spectinomycin 50 ppm, and kanamycin 50 ppm.

TABLE 8 Strains MCM433, 437, and 438 Host Strain Parent OriginIntegrated Plasmid(s) Markers MCM433 MCM331 BL21(DE3) gi1.2KKDyIpCLUpperHGS2 cmp5, spec50 MCM437 MCM433 BL21(DE3) gi1.2KKDyIpCLUpperHGS2 cmp5, pTrcHis2B kan spec50. (MCM94) kan50 MCM438 MCM433BL21(DE3) gi1.2KKDyI pCLUpperHGS2 cmp5, pTrcKudzuMVK(mazei) spec50.MCM376 kan50III. Cell FermentationMedium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 13.6 g, KH₂PO₄ 13.6g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate0.3 g, (NH₄)₂SO₄ 3.2 g, yeas extract 1 g, and 1000× Trace Metal Solution1 ml. All of the components were added together and dissolved in diH₂O.The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought tovolume. Media was filter sterilized with a 0.22 micron filter. Glucose5.0 g and antibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

1000× Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Each component wasdissolved one at a time in DI H₂O, pH to 3.0 with HCl/NaOH, then broughtto volume and filter sterilized with a 0.22 micron filter.

Strains:

The MCM343 strain is BL21 (DE3) E. coli cells containing the uppermevalonic acid (MVA) pathway (pCL PtrcUpperPathway encoding E. faecalismvaE and mvaS), the integrated lower MVA pathway (gi1.2KKDyI encoding S.cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase), and isoprene synthasefrom Kudzu (pTrcKudzu). This strain has low MVK polypeptide activity andhigh isoprene synthase polypeptide activity.

The MCM401 strain is BL21 (DE3) E. coli cells containing the upper MVApathway (pCL PtrcUpperPathway), the integrated lower MVA pathway(gi1.2KKDyI), and high expression of MVK from M. mazei and IS from Kudzu(pTrcKudzuMVK(M. mazei). This strain has high MVK polypeptide activityand high isoprene synthase polypeptide activity.

The MCM437 strain is BL21 (DE3) E. coli cells containing the upper MVApathway and low expression of IS from Kudzu (pCLPtrcUpperPathwayHGS2),the integrated lower MVA pathway (gi1.2KKDyI), and a control plasmidconferring kanamycin resistance (so that the growth media was identicalin all cases). This strain has low MVK polypeptide activity and lowisoprene synthase.

The MCM438 strain is BL21 (DE3) E. coli cells containing the upper MVApathway and low expression of IS from Kudzu (pCLPtrcUpperPathwayHGS2),the integrated lower MVA pathway (gi1.2KKDyI), and strong expression ofM. mazei MVK (M. mazei MVK in pET200). This strain has high MVKpolypeptide activity and low isoprene synthase polypeptide activity.

Isoprene production was analyzed by growing the strains in a Cellerator™from MicroReactor Technologies, Inc. The working volume in each of the24 wells was 4.5 mL. The temperature was maintained at 30° C., the pHsetpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitationrate was 800 rpm. An inoculum of E. coli strain taken from a frozen vialwas streaked onto an LB broth agar plate (with antibiotics) andincubated at 30° C. A single colony was inoculated into media withantibiotics and grown overnight. The bacteria were diluted into 4.5 mLof media with antibiotics to reach an optical density of 0.05 measuredat 550 nm.

Off-gas analysis of isoprene was performed using a gaschromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay.Sample preparation was as follows: 100 μL of whole broth was placed in asealed GC vial and incubated at 30° C. for a fixed time of 30 minutes.Following a heat kill step, consisting of incubation at 70° C. for 5minutes, the sample was loaded on the GC.

Optical density (OD) at a wavelength of 550 nm was obtained using amicroplate reader (Spectramax) during the course of the run. Specificproductivity was obtained by dividing the isoprene concentration (μg/L)by the OD reading. Samples were taken at three time points for each ofthe 24-wells over the course of the mini-fermentations. There were sixreplicates for each strain (4 strains×6 wells/strain).

Specific productivity of isoprene from a strain expressing the fullmevalonic acid pathway and Kudzu isoprene synthase at low levels(MCM437) was compared to a strain that in addition over-expressed MVKfrom M. mazei and Kudzu isoprene synthase (MCM401), as well as strainsthat either over-expressed just MVK (MCM438), or just Kudzu isoprenesynthase (MCM343). The bacteria were grown under identical conditions indefined media with glucose as a carbon source in mini-fermentations.Induction of isoprene production was achieved by adding IPTG to a finalconcentration of 200 μM at the start of the run. Headspace measurementsover time (FIG. 120) revealed that the strain over-expressing both MVKand isoprene synthase (MCM401) had higher specific productivity ofisoprene compared to the strain over-expressing just MVK (MCM438) orjust Kudzu isoprene synthase (MCM343). The strain with low activities ofboth MVK and Kudzu isoprene synthase (MCM437) had the lowest specificproductivity of isoprene overall.

IV. Determination of Isoprene Synthase Polypeptide Activity andVolumetric Productivity in Fermentation Runs.

Strain MCM401 that overexpresses both M. mazei MVK and isoprene synthasehad a greater maximum volumetric productivity for isoprene than eitherstrain MC343 or strain MCM127 that do not express M. mazei MVK.

(i). Isoprene Synthase DMAPP Activity from Lysate Protocol

For this assay, the following reagents were used: 50% glycerol in PEBcontaining 1 mg/mL lysozyme (Sigma) and 0.1 mg/mL DNAaseI (Sigma). 1 mLof fermentation broth was mixed with 1 mL of 50% glycerol in PEBcontaining 1 mg lysozyme and 0.1 mg DNAaseI. The mixture is passedthrough the french press one time. 25 μL of the mixture is then used forthe DMAPP assay. The DMAPP assay contained the following components:

DMAPP Assay

-   25 μL lysate mixture-   5 μL MgCl₂ (1 M)-   5 μL DMAPP (100 mM)-   65 μL 50 mM Tris pH 8-   Total volume: 100 μL

The reaction is performed at 30° C. for 15 minutes in a gas tight 1.8 mLGC tube. Reactions are terminated by the addition of 100 μL 250 mM EDTA(pH 8).

The active protein concentration was measured using Equation 14.mg/mL active isoprene synthase=(Dilution factor)*X ug/L(DMAPP Assayreading)*0.0705/294(specific activity from 14-L)or 0.0002397*Xug/L  Equation 14

The volumetric productivity was measured using Equation 15.mg/L/h isoprene=(dilution factor)*0.288*X ug/L(DMAPP Assayreading)  Equation 15

The maximum in vitro isoprene synthase polypeptide activity was comparedwith the maximum volumetric productivity for strains MCM401, MC343, andMCM127 (FIG. 136).

Example 7 Exemplary Methods for Producing Isoprene: IsopreneFermentation from E. coli Expressing Genes from the Mevalonic AcidPathway and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH2O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andbrought to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibioticswere added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Trace Metal Solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO4*5H₂O100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolvedone at a time in Di H2O, pH to 3.0 with HCl/NaOH, then brought to volumeand filter sterilized with 0.22 micron filter.

I. MCM343 High Titer: Isoprene Fermentation from E. coli ExpressingGenes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture atthe 15-L Scale

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the gi1.2 integrated lower MVA pathway and the pCLPtrcUpperMVA and pTrcKudzu plasmids. This experiment was carried out tomonitor isoprene formation from glucose at the desired fermentation pH7.0 and temperature 30° C. An inoculum of E. coli strain taken from afrozen vial was streaked onto an LB broth agar plate (with antibiotics)and incubated at 37° C. A single colony was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 58 hour fermentation was 4.5 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 98 uM when the carbon dioxideevolution rate reached 25 mmol/L/hr (OD₅₅₀=9). The OD₅₅₀ profile withinthe bioreactor over time is shown in FIG. 112C. The isoprene level inthe off gas from the bioreactor was determined using a Hiden massspectrometer. The isoprene titer increased over the course of thefermentation to a final value of 1.6 g/L (FIG. 112D). The total amountof isoprene produced during the 58 hour fermentation was 17.9 g and thetime course of production is shown in FIG. 112E. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 0.8%. The weight percent yield of isoprene from glucose was 0.4%.

II. MCM127: Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 5-Lbioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 43 hour fermentation was 1.4 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 23 uM when the carbon dioxideevolution rate reached 25 mmol/L/hr (OD₅₅₀=129). The OD₅₅₀ profilewithin the bioreactor over time is shown in FIG. 112F. The isoprenelevel in the off gas from the bioreactor was determined as previouslydescribed by measuring isoprene concentrations in the offgas by GC. Theisoprene titer increased over the course of the fermentation to a finalvalue of 0.4 g/L (FIG. 112G). The total amount of isoprene producedduring the 43 hour fermentation was 3.0 g and the time course ofproduction is shown in FIG. 112H. The molar yield of utilized carbonthat went into producing isoprene during fermentation was 0.5%. Theweight percent yield of isoprene from glucose was 0.3%.

III. dxr Knock-Out Strain: Isoprene Fermentation from E. coli ExpressingGenes from the Mevalonic Acid Pathway and Grown in Fed-Batch Culture atthe 15-L Scale.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells (Δdxr) containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids.This experiment was carried out to monitor isoprene formation fromglucose at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 15-L bioreactor containing an initial volume of 5-L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 43 hour fermentation was 1.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 uM when the optical density at 550nm (OD₅₅₀) reached a value of 8. The IPTG concentration was raised to 40uM when OD₅₅₀ reached 140. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 112I. The isoprene level in the off gas from thebioreactor was determined as previously described (GC of offgassamples). The isoprene titer increased over the course of thefermentation to a final value of 0.9 g/L (FIG. 112J). The total amountof isoprene produced during the 43 hour fermentation was 6.0 g and thetime course of production is shown in FIG. 112K. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 0.8%. The weight percent yield of isoprene from glucose was 0.4%.

(i) Construction of the dxr Mutant in E. coli

To generate a deletion of dxr (1-deoxy-D-xylulose 5-phosphatereductoisomerase), the enzyme that encodes the first committed step inthe deoxy-xylulose-phosphate (DXP) pathway in Escherichia coli, theGeneBridges Quick & Easy E. coli Gene Deletion Kit (GB) was usedaccording to the manufacturer's recommended protocol. Briefly, GBinsertion cassettes encoding either kanamycin (FRT-PGK-gb2-neo-FRT) orchloramphenicol (FRT-cm-FRT) resistance were PCR amplified using primersGBdxr1 and GBdxr2 (see below for primer sequences and cyclingparameters). PCR products of the correct size (for the respective GBinsertion cassette) were pooled, purified (Qiagen) and diluted to aconcentration of approximately 300 ng/μl. The deletion of dxr was thencarried out according to the protocol described in the GB manual. Allreplicating plasmids were introduced into E. coli strains viaelectroporation using standard molecular biology techniques (see Table16 below for a complete strain list). LB medium containing ampicillin(50 μg/ml) and spectinomycin (50 μg/ml) was inoculated with E. colistrains (DW13 or DW38) harboring the pRed/ET plasmid (encodingampicillin/carbenicillin resistance) and pCL Ptrc(minus lacO) KKDyI(from Edwin Lee, encoding spectinomycin resistance). These strainscarried pCL Ptrc(minus lacO) KKDyI (see (iv) below) so that E. coli, inthe absence of a functional DXP pathway, could convert mevalonic acid(MVA) through the MVA lower pathway to IPP/DMAPP as a source for alllower isoprenoid molecules. Cultures were grown overnight at 30° C. anddiluted to an OD₆₀₀ of approximately 0.2 in 5 ml total volume withantibiotics the next morning. After several hours of growth at 30° C.,strains were shifted to 37° C. and L-arabinose was added at aconcentration of 0.4%. After 1 hour of induction, cells were washedmultiple times in ice cold H₂O, and approximately 700 ng of the purifiedPCR product (described above) for each GB insertion template wasintroduced via electroporation (using standard techniques). Cells wererecovered for 3 hours at 37° C. in LB with 1 mM MVA with no antibiotics,and then plated onto selective LB medium (MVA 1 mM and spectinomycin 50μg/ml, with either kanamycin 15 μg/ml or chloramphenicol 25 μg/ml). Thenext day, positive colonies were tested by PCR, using the dxrTest1 anddxrTest2 primers, with either GBprimer2 or GBprimerDW (i.e. GB3, seeFIG. 112M), respectively (see Table 16). Colonies that tested positivewith these primer combinations were then tested for sensitivity to MVAat varying concentrations. FIG. 112J shows that in the absence of MVA,dxr deletion strains are unable to grow, whereas in the presence of 1 mMMVA, growth is robust. FIG. 112N also shows that at a concentration of10 mM MVA, growth of dxr deletion strains appears to be inhibited, mostlikely because of the accumulation of isoprenoid molecules. To generatestrain DW48, strain DW43 was electroporated with plasmids MCM82 (Sp) andMCM118 (Kan), which harbor the entire MVA pathway and HGS. Since MVA wasomitted from recovery and on the selective plate (LB with Sp μg/ml andKan μg/ml), strain DW48 was forced to lose plasmid pCL Ptrc(minus lacO)KKDyI and gain MCM82, which contains the MVA upper pathway. Thus, onlycells harboring the entire MVA pathway to convert acetyl-CoA toIPP/DMAPP and lower isoprenoids were able to grow without exogenous MVA.

(ii) PCR Cycling Parameters

The Herculase II (Stratagene) DNA polymerase enzyme was used foramplification of all GB templates with oligonucleotide primer pairs at aconcentration of 0.4 μM each in 50 μl total volume/reaction according tothe manufacturer's protocol. All PCR products for generating dxrdeletion strains via GB were of the expected size: approximately 1.6 kb(kanamycin), and 1.5 kb (chloramphenicol).

To test GB insertions at the dxr locus, illustra PuReTaq Ready-To-Go™PCR Beads (GE Healthcare) were used with oligonucleotide primer pairs ata concentration of 0.4 μM each in 25 μl total volume/reaction.

-   1) 95° C.—4 min-   2) 95° C.—20 sec-   3) 55° C.—20 sec (52° C. for Beads)-   4) 72° C.—2 min (30 sec for Beads)-   5 cycles of steps 2 through 4-   5) 95° C.—20 sec-   6) 58° C.—20 sec (55° C. for Beads)-   7) 72° C.—2 min (30 sec for Beads)-   25 cycles of steps 5 through 7-   72° C.—10 min-   4° C.—end

TABLE 16 PCR primers, plasmids, and Strains Primer Name Sequence (5′to 3′) Purpose GBdxr1 GGCTGGCGGCGTTTTGCTTTTTATTdxr knock out GB-Forward primer for CTGTCTCAACTCTGGATGTTTCATGall templates AATTAACCCTCACTAAAGGGCG (SEQ ID NO: 130) GBdxr2AAGCCCTACGCTAACAAATAGCGC dxr knock out GB-Reverse primer forGACTCTCTGTAGCCGGATTATCCTC all GB templates ATAATACGACTCACTATAGGGCTC(SEQ ID NO: 131) dxrTest1 ACGCCGCTCAGTAGATCCTTGCGG 5′of 50 bp homology region (in GBdxr1) AT used for GB knock-out(SEQ ID NO: 132) dxrTest2 CTACTTACGATCAGATGGCGCAGA 3′of 50 bp homology region (in GBdxr2) CTA used for GB knock-out(SEQ ID NO: 133) GBprimer2 CGAGACTAGTGAGACGTGCTACGB test primer all cassettes-amplifies (SEQ ID NO: 134) towards 5′ endGBprimerDW AAAGACCGACCAAGCGACGTCTGAGB test primer all cassettes-amplifies (SEQ ID NO: 135) towards 3′ endPlasmid Resistance purpose pCL Ptrc (minus Spectinomycin (sp)Lower MVA pathway for conversion of lacO) KKDyIMVA to IPP/DMAPP-lower isoprenoids FRT-cm-FRT Chloramphenicol (GBchlor)GB template-chloramphenicol FRT-PGK-gb2- Kanamycin (GBkan)GB template-kanamycin neo-FRT pRedET Ampicillin (amp)GB L-arabinose inducible expression of Red/ET proteins MCM82Spectinomycin (sp) Upper MVA pathway MCM118 Kanamycin (kan)Lower MVA pathway + HGS Strain Genotype purpose DW13MG1655 with pCL Ptrc (minus lacO) Parent strain of dxr deletion-has KKDyI and pRedET, sp, amp entire MVA lower pathway DW23MG1655 Δdxr::GBkan with pCL dxr delection (kan) in MG1655Ptrc (minus lacO) KKDyI, kan, sp DW28 MG1655 Δdxr::GBchlor with pCLdxr delection (chlor) in MG1655 Ptrc (minus lacO) KKDyI, chlor, sp DW38BL21 DE3 (Invitrogen) with pCL Parent strain of dxr deletion-has Ptrc (minus lacO) KKDyI and pRedET, entire MVA lower pathway sp, ampDW43 BL21 DE3 Δdxr::GBchlor with pCL dxr delection (chlor) in BL21 DE3Ptrc (minus lacO) KKDyI, chlor, sp DW48BL21 DE3 Δdxr::GBchlor with MCM82 dxr delection (chlor) in BL21 DE3 withand MCM118, sp, kan entire MVA pathway-requires no MVA(iii) Construction of MCM184-pCL Ptrc(minus lacO) UpperPathway

Plasmid MCM82 was mutagenized using the Stratagene QuikChange XL II kit.A reaction consisting of 10 uL buffer, 1 uL 100 ng/uL MCM82 DNA, 2.5 uL10 uM primer MCM63 (SEQ ID NO:123), 2.5 uL 10 uM primer MCM64 (SEQ IDNO:124), 2 uL dNTP mix, 6 uL QuikSolution, 76 uL ddH2O and 2 uLpolymerase was combined and aliquotted to four PCR tubes. Tubes werecycled in columns 1, 4, 7 and 12 of a BioRad 96-well gradient blockusing 1×95C for 1 minute, 18×95° C. for 50 seconds, 60-65° C. for 50seconds, 68° C. for 10 minutes), 1×68° C. for 7 minutes, 1×4° C. untilcool. 1 uL DpnI was added and reactions were incubated at 37° C. for 2hr and then frozen overnight at −20° C. 5 uL was transformed intoInvitrogen TOP10 OneShot cells according to the manufacturer's protocol.Transformants were selected on LA+50 ppm Spectinomycin. Several colonieswere cultured in LB+spectinomycin50 and then used for plasmidpurification. Clone 2 from reaction 3 (column 7 from gradient block PCR)had the expected sequence and was frozen as MCM184.

(iv) Construction of pCL Ptrc(ΔlacO) KKDyI (as Referred to as pCL Ptrc(Minus lacO) KKDyI or pCL Ptrc (Minus lacO) Lower Pathway)

Plasmid MCM184 (pCL Ptrc(minus lacO) UpperPathway) was digestedsequentially with SacI and PstI restriction endonucleases to remove theUpper MVA Pathway. A reaction consisting of 8 uL MCM184 (80 ng/uL), 3 ulRoche 10× Buffer A, 2 uL SacI restriction endonuclease, and 17 uL ddH₂Owas prepared and incubated at 37° C. for 2 hours. The SacI restrictionendonuclease was then inactivated by heating at 65° C. for 20 minutes.The DNA fragment was then purified by using a Qiagen PCR Purificationcolumn per manufacturer's protocol. The DNA fragment was then elutedfrom the column with a volume of 34 uL ddH₂O. The next (sequential)restriction digest reaction consisted of the 34 uL SacI digested eluant,4 uL Roche 10× Buffer H, and 2 uL PstI restriction endonuclease. Thereaction was incubated at 37° C. for 2 hours before being heatinactivated at 65° C. for 20 minutes. A dephosphorylation step was thenperformed by addition of 4.7 uL Roche 10× Shrimp Alkaline Phosphatase(SAP) buffer), and 2 uL SAP enzyme. The reaction was then incubated at37° C. for 1 hour. The digested MCM184 vector backbone was thenseparated from the Upper MVA Pathway DNA fragment by electrophoresis ona 1.2% E-gel (Invitrogen).

The Lower MVA Pathway fragment (KKDyI) was digested sequentially withSacI and PstI restriction endonucleases from plasmid MCM107. A reactionconsisting of 2 uL MCM107 (375 ng/uL), 3 uL Roche 10× Buffer A, 2 uLSacI restriction endonuclease, and 23 uL ddH₂O was prepared andincubated at 37° C. for 3 hours. The SacI restriction endonuclease wasthen inactivated by heating at 65° C. for 20 minutes. The DNA fragmentwas then purified by using a Qiagen PCR Purification column permanufacturer's protocol. The DNA fragment was then eluted from thecolumn with a volume of 34 uL ddH₂O. The sequential digest reactionconsisted of the 34 uL SacI digested eluant, 4 uL Roche 10× Buffer H,and 2 uL PstI restriction endonuclease. The reaction was incubated at37° C. for 2 hours before being heat inactivated at 65° C. for 20minutes. The digested KKDyI fragment was then separated from the MCM107vector backbone by electrophoresis on a 1.2% E-gel (Invitrogen).

A ligation reaction consisting of 3 uL MCM184 vector backbone, 6 uLKKDyI DNA fragment, 2 uL New England Biolabs (NEB) 10× T4 DNA LigaseBuffer, 1 ul T4 DNA ligase, and 8 uL ddH₂O were incubated at roomtemperature for 20 minutes. The ligation reaction was then transformedinto TOP10 chemically competent E. coli cells (Invitrogen) permanufacturer's protocol and plated on LA+50 ppm spectinomycin plates. Toconfirm that transformants had correct sized insert fragment, a PCRscreen was performed. 50 uL ddH₂O was inoculated with individualcolonies from the transformation, boiled at 95° C. for 5 minutes, andmicrocentrifuged for 5 minutes to pellet cellular debri. PCR wasperformed using PuReTaq Ready-To-Go PCR beads (GE Healthcare).Individual reaction tubes contained 1 uL of boiled cell lysate, 1 uL 10uM primer EL-976 (SEQ ID NO:126), 1 uL 10 uM primer EL-977 (SEQ IDNO:127), and 22 uL ddH₂O. PCR tubes were cycled 1×95° C. for 1 minute,30× (95° C. for 30 seconds, 53° C. for 30 seconds, 72° C. for 45seconds), 1×72° C. for 2 minutes. The PCR products were then analyzed ona 1.2% E-gel for an 840 bp fragment. Clones #2, #3, and #4 werecontained the correct sized fragments and were DNA sequenced usingprimers EL-976 (SEQ ID NO:126) and EL-978 (SEQ ID NO:128). DNAsequencing confirmation showed that all 3 were correct.

Example 8 Metabolite Analysis, Growth Inhibition, and FeedbackInhibition

I. Metabolite Extraction from E. coli. Sampled from 14-L Fermentors.

The metabolism of bacterial cells grown in fermentors was rapidlyinactivated by withdrawing approximately 4 mL of culture into a tubefilled with 8 mL of dry ice-cold methanol. The resulting samples wereweighed to calculate the amount of sampled broth and then put into −80°C. for storage until further analysis. For metabolite extraction andconcentration, 1.5 to 4.0 mL aliquots of cell suspension were dilutedwith methanol/ammonium acetate buffer (5 mM, pH=8.0) mixture (6:1, v/v)to a final volume of 6 mL, and cell debris was pelleted by a 5 minutecentrifugation. The supernatant was collected and loaded onto aStrata-X-AW column (Phenomenex) containing 30 mg of sorbent thatselectively retains strong organic acids. The pellet was extracted twomore times, first with 3 mL of the methanol/ammonium acetate buffer (5mM, pH=8.0) mixture (6:1 v/v), and then with 6 mL of methanol/ammoniumacetate buffer (5 mM, pH=8.0) mixture (1:1 v/v). Both times the cellswere pelleted by centrifugation, and the resulting supernatants wereconsecutively loaded onto the same Strata-X-AW column. During theextraction-centrifugation, samples with cells were kept below 4° C. tominimize degradation of metabolites. After washing the columns with 1 mLof water and 1 mL of methanol, metabolites of interest were eluted fromthe columns first with 0.3 mL of concentrated NH₄OH/methanol (1:14, v/v)mixture and then with 0.3 mL of concentrated NH₄OH/methanol/water(1:12:2, v/v) mixture. The resulting eluant was neutralized by adding 20μL of glacial acetic acid, and then cleared by centrifugation in amicrocentrifuge.

II. Metabolite Extraction from E. coli. Grown in Shake Flasks.

To extract metabolites from shake flask-grown E. coli, methanol-quenchedcells were pelleted by centrifugation, and the resulting supernatant wasloaded onto Strata-X-AW anion exchange column (Phenomenex) containing 30mg of sorbent. The pellet was re-extracted twice with severalmilliliters of 50%, v/v, aqueous methanol containing 20% ammoniumbicarbonate buffer (pH=8.0) and then with 75%, v/v, aqueousbicarbonate-buffered methanol. After each extraction, cell debris waspelleted by centrifugation, and the supernatant was consecutively loadedonto the same anion exchange columns. During the extraction andcentrifugation steps, the samples were kept at below +4° C. Prior tometabolite elution, the columns were washed with water and methanol (1mL of each), and the analytes were eluted by adding 0.3 mL ofconcentrated NH₄OH/methanol (1:14, v/v) and then 0.3 mL of concentratedNH₄OH/water/methanol (1:2:12) mixtures. The eluant was neutralized with40 μL of glacial acetic acid and then cleared by centrifugation in amicrocentrifuge.

III. Metabolite Quantification

Analysis of metabolites was carried out using a Thermo Finnigan TSQsystem (Thermo Electron Corporation, San Jose, Calif.). All systemcontrol, data acquisition, and mass spectral data evaluation wereperformed using XCalibur and LCQuan software (Thermo Electron Corp). Forthe LC-ESI-MS/MS method, a chiral Nucleodex β-OH 5 μM HPLC column (200×4mm, Macherey-Nagel, Germany) was used with a CC 8/4 Nucleodex beta-OHguard cartridge. A mobile phase gradient (Table 9) was applied at a flowrate of 0.8 mL/min in which mobile phase A was MilliQ-grade water,mobile phase B was 100 mM ammonium acetate (SigmaUltra grade, Sigma)buffer (pH adjusted to 8.0 by ammonium hydroxide) in MilliQ-grade waterand mobile phase C was LCMS grade acetonitrile (Chromasolv, Riedel-deHaën). The column and sample tray temperatures were reduced to 5° C. and4° C., respectively. The injection volume was 10 or 20 μL. FIG. 121shows typical elution profiles of selected metabolites extracted from anisoprene-producing E. coli strain.

TABLE 9 HPLC gradient used to elute metabolites in the MVA pathway.Mobile phase, % B Time, A (100 mM ammonium C min (water) acetate, pH =8.0) (acetonitrile) 0.0 0.0 20.0 80.0 1.0 0.0 20.0 80.0 8.0 0.0 50.050.0 11.0 0.0 50.0 50.0 13.0 46.0 4.0 50.0 19.0 49.6 0.4 50.0 22.5 49.60.4 50.0 23.0 0.0 20.0 80.0 25.0 0.0 20.0 80.0

Mass detection was carried out using electrospray ionization in thenegative mode (ESI spray voltage of 2.5-3.0 kV and ion transfer tubetemperature of 390° C.). The following m/z values for precursor ionswere selected to detect the metabolites of interest in SRM mode: 245.0for IPP and DMAPP, 313.1 for GPP, 381.1 for FPP, 227.0 for MVP, and307.1 for MVPP. Concentrations of metabolites were determined based onthe integrated intensities of peaks generated by PO₃ ⁻ product ion(m/z=79.0). Calibration curves obtained by injection of standards (IPP,DMAPP, and GPP purchased from Sigma-Aldrich, and FPP purchased fromEchelon Biosciences Inc.) were used to calculate concentrations ofmetabolites in cell extracts. Concentrations of MVP and MVPP wereexpressed in arbitrary units because of the absence of commerciallyavailable standards. Intracellular concentrations of metabolites weredetermined based on the assumption that in 1 mL of the culture at OD=200the integrated volume of all cells is 50 μL.

IV. Intracellular Concentrations of Metabolites in the MCM401 Strain ofE. coli Containing MVK from M. mazei Under Different Levels of EnzymeExpression Induced by Adding IPTG to the Fermentors.

FIG. 122A-122F provide an example of intracellular concentrations ofmetabolites in the MCM401 strain of E. coli containing MVK from M. mazeiunder different levels of enzyme expression induced by adding IPTG tothe fermentors. Even though the final IPTG concentrations in all threefermentors were similar (˜200 μM), cell response was very differentdepending on the IPTG feeding scheme. A single-shot addition of a highdose of IPTG (FIGS. 122C and 1224F) caused an instant increase inisoprene production and early accumulation of a significant level ofMVPP. In contrast, concentrations of DMAPP, the immediate precursor ofisoprene, as well as GPP and FPP, the products of IPP and DMAPPcondensation, were low (below ˜0.2 mM). Intracellular concentrations ofIPP remained higher than the concentration of DMAPP during the analyzedfermentation period, indicating that DMAPP is synthesized from IPPslower than it is consumed in the isoprene biosynthesis reaction.

Although the maximum specific productivity of MCM401 cells reached aboutthe same level upon adding IPTG in two steps (˜100 μM each time; FIGS.122B and 122E), the amount of MVPP accumulated in cells by the end ofthe production period was lower than in the single IPTG shot experimentand the buildup of MVPP pool started only after the second portion ofIPTG was added to the fermentor. In both cases a decline in the isopreneproduction correlated with accumulation of MVP, which pool reached muchhigher concentrations in cells that had received two doses of IPTG.Moderate levels of IPP and DMAPP (˜0.4 mM) were detected in the lattercase around 30 hours of fermentation, which correlated in time with themaximum rate of isoprene biosynthesis by these cells. Notably,intracellular concentrations of GPP and FPP were low presumably due to avery high activity of the isoprene synthase.

Four IPTG shots of about 50 μM each resulted in the lowest specificproductivity of the MCM401 strain; however, under these conditions theculture continued to synthesize isoprene at a significant rate for alonger period of time (FIGS. 122A and 122D). The maximum intracellularlevels of IPP and DMAPP generally remained in the range of 0.2-0.4 mMduring the production period, and FPP raised to 1.0-1.5 mM in responseto the second 50 μM dose of IPTG. Notably, DMAPP concentration wasslightly higher than the concentration of IPP likely due to the factthat DMAPP conversion into isoprene occurred slower in this casecompared to the fermentations illustrated in FIGS. 122B, 122C, 122E, and122F, and FPP biosynthesis did not consume significant amounts of DMAPP.

V. Intracellular Concentrations of Metabolites in the MCM402 Strain ofE. coli Overexpressing MVK from Saccharomyces cerevisiae

FIGS. 127A and 127B illustrate the experiment with the MCM402 strain ofE. coli, containing overexpressed MVK from Saccharomyces cerevisiae. Asin the case with the MCM401 strain having MVK from M. mazei and grownunder similar IPTG induction conditions (4×50 μM shots), isopreneproduction started after the second dose of IPTG has been added to thefermentor, which coincided in time with rapid accumulation of DMAPP andIPP to relatively high levels (up to 1.8 mM of DMAPP) in the MCM402cells. However, in the MCM402 cells, the isoprene production periodremained very short correlating with the drop in DMAPP and IPP pools. Incontrast, FPP continued to accumulate up to the level of 2.6-3.5 mM evenwhen DMAPP and IPP concentrations dropped to below 1 mM. Therefore,parts IV and V of this example emphasize superior properties of MVK fromM. mazei as compared to yeast MVK.

VI. Intracellular Concentrations of Metabolites in the MCM343 Strain ofE. coli Expressing the Full Mevalonic Acid Pathway and Kudzu IsopreneSynthase (Without Overexpression of a Second Mevalonate Kinase)

FIGS. 128A and 128B depict changes in concentrations of selectedintermediates in the isoprenoid pathway in the course of fermentation ofMCM343 E. coli strain. This fermentation run was characterized by verylow specific productivity and barely detectable concentrations of mostof isoprenoid intermediates except for FPP, which intracellular levelreached 0.7 mM, after 100 μM IPTG was added to the cells. IPP and DMAPPwere detected shortly after the IPTG addition and then their leveldropped below the detection limit. No MVP or MVPP were detected duringthe fermentation.

VII. Safe and Maximal Metabolite Concentrations during IsopreneProduction Shake Flask Experiment with MCM127

A shake flask experiment with MCM127 was performed to investigate theaccumulation of key intermediates during strong induction of isopreneproduction. Strong induction of this strain resulted in growthinhibition most likely due to accumulation of toxic metabolicintermediates.

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 13.6 g, KH₂PO₄ 13.6g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g, 1000× Trace Metal Solution 1ml. All of the components were added together and dissolved in diH₂O.The pH was adjusted to 6.8 with ammonium hydroxide (30%) and brought tovolume. Medium was filter-sterilized with a 0.22 micron vacuum filter.Glucose was added to the medium to a final concentration of 0.5%.Antibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Medium):

1000× trace metal solution contained citric Acids*H₂O 40 g, MnSO₄*H₂O 30g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolvedone at a time in diH₂O, pH to 3.0 with HCl/NaOH, and then brought tovolume and filter sterilized with 0.22 micron filter.

Strain:

The MCM127 strain is BL21 (DE3) E. coli cells containing the uppermevalonic acid (MVA pathway (pCL Upper) and the lower MVA pathwayincluding isoprene synthase from kudzu (pTrcKKDyIkIS)

An inoculum of E. coli strain MCM127 taken from a frozen vial wasstreaked onto an LB broth agar plate (with antibiotics) and incubated at30° C. A single colony was inoculated into media containing glucose ascarbon source and grown overnight at 30° C. The bacteria were dilutedinto fermentation media to reach an optical density of 0.05 measured at550 nm. A total of 150 mL of culture was dispensed into two 500 mLflasks that were then shaken at 170 rpm in a 30° C. incubator. When thecultures reached an optical density (OD₆₀₀) of 0.5, one of the flaskswas induced with 150 μM isopropyl-beta-D-1-thiogalactopyranoside (IPTG).Samples of 20 mL from both the induced and non-induced culture weretaken approximately every half hour for metabolite analysis afterinduction. The samples were quickly quenched in equal volume of methanolcooled on dry ice. After centrifugation, supernatant was loaded on StataX-AW columns. The pellet was resuspended in 5 mL of Methanol-water (6:1,water contained 5 mM NH4Ac at pH=8.0), cell debris were separated bycentrifugation, and the supernatant was loaded on the Stata X-AWcolumns. Metabolites were eluted with 0.30 mL ethanol:conc NH4OH (14:1vol/vol), then with 0.3 mL methanol:water:conc NH4OH (12:2:1vol/vol/vol), finally pH was adjusted by adding 40 uL of glacial aceticacid. Extracted metabolites were analyzed by LCMS using a standardcyclodextrin column protocol. To increase sensitivity, only ionscorresponding to IPP, DMAPP, GPP, and FPP were detected. Injectionvolume was 20 uL/sample. Standards of all metabolites were used forcalibration.

Upon induction of the MCM127 with 150 μM IPTG, the bacteria continued togrow identical to the non-induced strain for approximately one and ahalf hour. After this, the induced culture began to show signs of growthinhibition (FIG. 112A). Key metabolites were measured during theexperiment and showed an increasing accumulation of FPP, GPP, DMAPP andIPP after induction. DMAPP and IPP only began to accumulate when theinduced bacteria first showed signs of growth inhibition (FIG. 112B).None of the mentioned intermediates were detected in measurable amountin the non-induced culture. The experiment demonstrates that E. coli cantolerate significant intracellular concentrations of GPP and FPP (Tables15A and 15B), while accumulation of DMAPP and IPP coincides with growthinhibition when cultures are grown in shake flasks. Data in Tables 15Aand 15B were from the 5.5 hr time point, where growth was still normalin the induced culture.

VIII. Growth Inhibition

i) Recovery of Mevalonic Acid from Fermentation Broth.

Mevalonic acid was obtained by a fed batch fermentation of Escherichiacoli strain, BL21 harboring an expression plasmid bearing the genes mvaSand mvaE from Enterococcus faecalis (U.S. Appl. Pub. No. 2005/0287655,which is incorporated by reference in its entirety, particularly withrespect to genes mvaS and mvaE). Fermentation of the strains was carriedout in fed batch fermentation mode in a minimal medium with a glucosefeed for 40 hours. Broth was harvested, mixed with diatomaceous earth(DE; Catalog #Celatom FW-12, American Tartaric Products Inc.), andfiltered under vacuum through a Buchner funnel fitted with a filter pad.The filtrate was sterile filtered through a 10,000 MWCO membrane.Mevalonic acid was converted to the lactone by acidification andrecovered by continuous organic solvent extraction; NMR analysisindicated a purity of 84%. All recovery steps are well known to thoseskilled in the art. When the free acid was required for experiments, theMVA lactone was hydrolyzed by the addition of 1 equivalent of base to asolution of lactone and allowed to stand for 1 hour prior to use. Thesterile filtered solution can be stored for extended time at 4° C.

ii) Growth Inhibition of Escherichia coli BL21 by the Accumulation ofMevalonate Diphosphate, Isopentenyl Diphosphate (IPP), and DimethylallylDiphosphate (DMAPP).

The purpose of this experiment was to determine the effect of theexpression of the proteins mevalonate kinase (MVK), phophomevalonatekinase (PMK), and diphosphomevalonate decarboxylase (MDD) on Escherichiacoli cultures.

E. coli BL21 cells bearing pTrcK, representing a plasmid expressing MVK,pTrcKK representing a plasmid expressing MVK plus PMK, and pTrcKKD,representing a plasmid expressing MVK plus PMK plus MDD were grown atapproximately 30° C. and 250 rpm in 250 mL flasks containing 25 mL ofTM3 medium (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 g MgSO₄*7H₂O) supplementedwith 1% glucose and 0.8 g/L Biospringer yeast extract (1% Yeast extractfinal). When OD600 reached 0.8 to 0.9, 5.8 mM mevalonic acid was addedto the cultures and incubation was continues for an additional 5 hours.OD₆₀₀ measurements were taken, and the cultures were sampled formetabolite analysis at 2 hours post MVA addition. Samples were collectedinto 100% MeOH prechilled in dry ice in a ratio of 1:1. Samples werestored at −80° C. until analyzed as follows. The methanol-quenched cellswere pelleted by centrifugation and the resulting supernatant was loadedonto Strata-X-AW anion exchange column (Phenomenex) containing 30 mg ofsorbent. The pellet was reextracted twice with several milliliters of50%, v/v, aqueous methanol containing 20% ammonium bicarbonate buffer(pH=8.0) and then with 75%, v/v, aqueous bicarbonate-buffered methanol.After each extraction, cell debris were pelleted by centrifugation andthe supernatant was consecutively loaded onto the same anion exchangecolumns. During the extraction and centrifugation steps, the sampleswere kept at below +4° C. Prior to metabolite elution, the columns werewashed with water and methanol (1 mL of each) and the analytes wereeluted by adding 0.3 mL of concentrated NH₄OH/methanol (1:14, v/v) andthen 0.3 mL of concentrated NH₄OH/water/methanol (1:2:12) mixtures. Theeluant was neutralized with 40 μL of glacial acetic acid and thencleared by centrifugation in microcentrifuge. Analysis of metabolites inthese samples is as described above.

As is shown in FIG. 129, inhibition of growth was evident when theenzymes MVK and PMK are expressed (strain #7); additional inhibition isobserved when MDD is added to the cloned pathway (strain #6). No growthinhibition was observed when MVK was the only enzyme expressed (strain#5). Analysis of MVA concentration at the time of collection of samplessuggests that strain with MVK plus PMK plus MDD consumed 2.9 mM MVAwhile the other two strains consume lower quantities. The culturecarrying MVK and PMV showed about 30 and 60-fold higher levels,respectively, of phosphomevalonate and diphosphomevalonate compared tothe strain carrying MVK, PMK, and MDD. The latter strain accumulatedsurprisingly high levels of IPP and DMAPP on the order of 40 mM IPP and320 uM DMAPP when calculated as an intracellular concentration. Thesemeasurements were conducted on whole cell broth; thus, some of themetabolites may have been excreted by the cells. While not intending tobe bound by any particular theory, it is believed that the observedgrowth inhibition is due to the accumulation of one or more of thesemetabolites. A goal is therefore to achieve a pathway enzyme balance tominimize the accumulation of these metabolites for the relief of growthinhibition.

IX. Feedback Inhibition

i) Methods and General Procedures

Geranyl-pyrophosphate (GPP), farnesyl-pyrophosphate (FPP), adenosinetriphosphate (ATP), phosphoenolpyruvate (PEP), NADH, magnesium chloride,sodium chloride, Tris, HEPES, DNase I, and lysozyme were purchased fromSigma. Dithiothreitol (DTT) was purchased from Fluka. Lactatedehydrogenase was purchased from Calbiochem and pyruvate kinase waspurchased from MD biomedicals. All columns used in purification wereobtained from GE healthcare. Purity was analyzed by 4-12% SDS-Page gelelectropheresis using precast gels and reagents purchased fromInvitrogen. Protein concentration was determined by UV-absorbance at 280nm using the following conversion factors: 0.597 OD/mg/mL for yeastmevalonate kinase and 0.343 OD/mg/mL for M. mazei mevalonate kinase(these were obtained using ExPASy ProtParam tool). Kinetics wereperformed using SpectraMax 190 platereader (Molecular Devices). Allkinetic data were analyzed using Kaleidagraph 4.0 graphing program fromSynergy software. Purified mevalonate was obtained using standardmethods.

ii) Expression and Purification of Yeast Mevalonate Kinase and M. mazeiMevalonate Kinase

Yeast and M. mazei mevalonate kinases were expressed as follows. E. colistrain MCM376 containing yeast MVK was grown at 37° C. in 2×1-L of LBmedia containing 50 mg/L carbenicillin and 30 mg/L chrolamphenicol.Cells were induced with 200 μM IPTG at OD₆₀₀=0.6-0.8. E. coli strainMD08-MVK containing M. mazei MVK was grown at 30° C. in 1-L of Terrificbroth with 50 mg/L kannamycin and 30 mg/L chloramphenicol. Cells wereinduced with 500 uM IPTG at OD₆₀₀=0.5. Identical harvest andpurification procedures were used for yeast and M. mazei mevalonatekinases. Cells were harvested by centrifugation approximately 15 hoursafter induction. Pelleted cells were resuspended in 15 mL Ni-bindingbuffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole,pH 8.0) and containing ˜1 mg/mL lysozyme and ˜0.1 mg/mL DNase I andfrench-pressed two times at 20,000 psi. Lysate was then centrifuged at229,000×g for one hour. Supernatant was loaded onto a Hi Trap IMAC HPcolumn charged with NiSO₄ and equilibrated with Ni-binding buffer.Column was washed with 10 column volumes of Ni-binding buffer. Yeast andM. mazei mevalonate kinases were eluted with a 0.02-0.5 M gradient ofimidizole. The buffer of fractions containing mevalonate kinase wereexchanged with 50 mM HEPES, 50 mM sodium chloride, pH 7.4, containing 1mM DTT using a Hi Prep 26/10 desalting column. Following desalting,mevalonate kinases were further purified over an anion exchange Hi TrapQ HP column. The column was washed with 50 mM Tris, 0.05 M sodiumchloride pH 7.6 containing 1 mM DTT, and eluted with a 0.05-1.0 M saltgradient. Fractions containing mevalonate kinase were desalted aspreviously described to 50 mM HEPES, 50 mM sodium chloride, pH 7.4containing 1 mM DTT to yield >95% pure yeast mevalonate kinase and M.mazei mevalonate kinase (as determined by SDS-PAGE and coomasiestaining).

iii) Kinetics of Yeast Mevalonate Kinase and M. mazei Mevalonate Kinase

The catalytic activities of the mevalonate kinases were determined usinga modified protocol (Beytia et al., (J. Biol. Chem. 245, 5450, 1970,which is incorporated by reference in its entirety, particularly withrespect to kinase assays). The assay was performed in a 96-well plateformat (Costar #9017) with a final reaction volume of 100 μl. Eachreaction contained the following reagents: 0.4 mM PEP, 0.05 mM DTT, 0.32mM NADH, 1 mM MgCl₂, 4 units of LDH and 4 units of PK in 50 mM Tris, 50mM NaCl, pH 7.6. The K_(M) value for yeast mevalonate kinase at themevalonate binding site was determined by adding 5 mM ATP to thereaction to saturate the ATP binding site, followed by addition ofmevalonate at concentrations ranging from 5 mM to 0.039 mM. The reactionwas initiated with the addition of 10 nM (50.1 ng) purified mevalonatekinase from yeast. The K_(M) value for yeast mevalonate kinase at theATP binding site was similarly determined, by saturating with 5 mMmevalonate and titrating ATP at concentrations ranging from 5 mM to0.039 mM. The K_(M) values for M. mazei mevalonate kinase weredetermined using the same procedure with the following exceptions:substrate concentrations ranged from 0 mM to 5 mM, and the reaction wasinitiated by adding 80 nM (0.25 μg) purified mevalonate kinase from M.mazei. Reactions were monitored by a decrease in absorbance at 340 nm.The concentration of NADH was plotted against time to determine the rateof the reactions. Units of absorbance were converted to μM NADH using aconversion factor determined from the difference in absorbance at 340 nmof 320 μM NADH and 320 μM fully oxidized NADH (NAD⁺) divided by the NADHconcentration (320 μM). Reactions were conducted at 30° C. and data werecollected every 10-15 seconds continuously over the course of thereactions.

Protein inhibition studies were performed using various concentrationsof terpenyl diphosphates.

iv) Yeast and M. mazei Mevalonate Kinase Kinetic Properties andInhibition Results

Kinetic studies were conducted using yeast mevalonate kinase and M.mazei mevalonate kinase. The K_(Mapp) of yeast mevalonate kinase wasdetermined to be 714±49 μM for the ATP binding site and 131±8 μM for themevalonate binding site with a turnover number (k_(cat)) of 38±5 s⁻¹(FIGS. 58A and 58B). The K_(Mapp) of M. mazei mevalonate kinase wasdetermined to be 464±12 μM for the ATP binding site and 68±4 μM for themevalonate binding site with k_(cat) of 4.3±0.2 s⁻¹ (FIGS. 58C and 58D).

Inhibition studies were performed using DMAPP, GPP, and FPP.Lineweaver-Burke plots of the inhibition studies demonstrate that theinhibition of yeast mevalonate kinase is competitive with respect to ATPand uncompetitive with respect to mevalonate (FIGS. 96A and 96B).Therefore, the inhibition constants for the ATP and mevalonate siteswere calculated by determining the IC₅₀ value followed by conversion tothe K_(i) value with Equation 16:

$\begin{matrix}{K_{i} = {\frac{{IC}_{50}}{1 + \frac{\lbrack S\rbrack}{K_{M}}}.}} & {{Equation}\mspace{14mu} 16}\end{matrix}$

The K_(i)s of DMAPP, GPP, and FPP for yeast mevalonate kinaseATP-binding site were determined to be 33.2 μM, 153.3 nM, and 138.5 nM,respectively (FIGS. 97A-97C). The K_(i)s of DMAPP, GPP, and FPP foryeast mevalonate kinase mevalonate-binding site were determined to be394.6 μM, 2.54 μM, and 2.98 μM, respectively (FIGS. 98A-98C).

M. mazei mevalonate kinase was not inhibited at concentrations of DMAPPup to 5 mM and concentrations of GPP and FPP up to 100 μM.

v) Diphosphomevalonate and Isopentyl Phosphate Inhibition of Yeast MVK,Streptococcus pneumoniae MVK, and Methanosarcina MVK

This experiment investigates the inhibitory effect ofdiphosphomevalonate and isopentyl monophosphate (IP) on MVK activityusing MVK enzymes from S. pneumoniae, yeast, and M. mazei, respectively.Inhibition of MVK by diphosphomevalonate and IP was shown using a twoenzyme system (Andreassi et al., Biochemistry, 43:16461-66, 2004, whichis incorporated by reference in its entirety with particular emphasis ondetermination of inhibition of MVK by diphosphomevalonate). Allreactions were performed in 96-well plates and were monitored byabsorbance at 386 nm on a Molecular Devices Spectramax 190 UV-Vis96-well spectrophotometer. All experiments were run as 100 μL reactionsat 30° C. and contained 5 mM ATP, 3 mM MgCl₂, 2.9 mM NADH, 0.7 mMR-mevalonate (Genencor), 4 mM phosphoenolpyruvate, 10 U lactatedehydrogenase (MP Biomedicals LLC), 10 U pyruvate kinase (MP BiomedicalsLLC), and 1 mM DTT. All chemical reagents were purchased from Sigmaunless otherwise specified. Yeast and M. mazei MVK were obtained asdescribed above and S. pneumoniae MVK and yeast phosphomevalonate kinase(PMK) were obtained as previously described (Andreassi et al.,Biochemistry, 43:16461-66, 2004) from pDW02 in pET200D MVK (FIG. 142).

When 1 μM S. pneumoniae MVK and 1 μM PMK are incubated with mevalonateand all essential co-factors, the reaction does not progress at the samerate as a reaction containing only S. pneumoniae MVK (FIG. 140A).However, the reaction proceeds to completion when PMK is added to theMVK-only reaction after that half of the reaction is complete (i.e. theproduction of phosphomevalonate), indicating that production ofdiphosphomevalonate inhibits S. pneumoniae MVK (FIG. 140B). Conversely,a reaction containing 1 μM Yeast MVK proceeds to completion regardlessof whether 1 μM PMK is present initially (FIG. 140C) or is added afteryeast MVK conversion of mevalonate to phosphomevalonate is complete(FIG. 140D), indicating yeast MVK is not inhibited bydiphosphomevalonate. Likewise, reactions containing 1 μM archaeal M.mazei MVK proceed to completion whether or not PMK is present initially(FIGS. 140E-140F) or added to the MVK-only reaction after that half ofthe reaction is complete (FIG. 140G).

Without being bound by theory, the archaeal mevalonate pathway has beenpostulated to contain an isopentyl monophosphate (IP) kinase thatcatalyzes the formation of isopentenyl diphosphate (IPP). Therefore,archaea may use an alternate mevalonate biosynthetic pathway to produceIPP and DMAPP. This putative pathway may contain a phosphomevalonatedecarboxylase that catalyzes the formation of IP from phosphomevalonate.When 100 μM of IPP was added to a reaction containing 1 μM M. mazei MVKand all reagents listed above, the reaction was not inhibited comparedto a control reaction containing all reagents listed above without theaddition of IP (FIG. 141).

S. pneumoniae MVK was inhibited by diphosphomevalonate according topreviously published results (FIGS. 140A-140B; Andreassi et al.,Biochemistry, 43:16461-66, 2004). Neither yeast MVK nor M. mazei MVK wasinhibited by diphosphomevalonate (FIGS. 140C-140G). M. mazei MVK was notinhibited by IP at concentrations up to 100 μM (FIG. 141). M. mazei MVKis thus the first described MVK that is not inhibited bydiphosphomevalonate, DMAPP, IP, GPP, or FPP.

Example 9 Production of Isoprene by E. coli Expressing the UpperMevalonic Acid (MVA) Pathway, the Integrated Lower MVA Pathway(gi1.2KKDyI), Mevalonate Kinase from Yeast, and Isoprene Synthase fromKudzu and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

Each liter of fermentation medium contained K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeastextract 0.5 g, and 1000× Modified Trace Metal Solution 1 ml. All of thecomponents were added together and dissolved in diH₂O. This solution wasautoclaved. The pH was adjusted to 7.0 with ammonium hydroxide (30%) andq.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics wereadded after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

1000× Modified Trace Metal Solution contained citric Acids*H₂O 40 g,MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg, and NaMoO₄*2H₂O 100 mg. Eachcomponent was dissolved one at a time in DI H₂O, pH to 3.0 withHCl/NaOH, then q.s. to volume and filter sterilized with a 0.22 micronfilter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCLPtrcUpperPathway encoding E. faecalis mvaE and mvaS), the integratedlower MVA pathway (gi1.2KKDyI encoding S. cerevisiae mevalonate kinase,mevalonate phosphate kinase, mevalonate pyrophosphate decarboxylase, andIPP isomerase), and high expression of mevalonate kinase from yeast andisoprene synthase from Kudzu (pTrcKudzuMVK(yeast)). This experiment wascarried out to monitor isoprene formation from glucose at the desiredfermentation pH 7.0 and temperature 30° C. An inoculum of E. coli straintaken from a frozen vial was streaked onto an LB broth agar plate (withantibiotics) and incubated at 37° C. A single colony was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 500 mL was used to innoculate 5-L of cell medium inthe 15-L bioreactor. The liquid volume increases throughout thefermentation (such as to approximately 10 liters).

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 1.6 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 54 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 87 uM when OD₅₅₀ reached 175.Additional IPTG additions raised the concentration to 122 uM atOD₅₅₀=180 and 157 uM at OD₅₅₀=185. The OD₅₅₀ profile within thebioreactor over time is shown in FIG. 123. The isoprene level in the offgas from the bioreactor was determined using a Hiden mass spectrometer.The isoprene titer increased over the course of the fermentation to afinal value of 6.4 g/L (FIG. 124). The total amount of isoprene producedduring the 54 hour fermentation was 44.6 g and the time course ofproduction is shown in FIG. 125. The molar yield of utilized carbon thatwent into producing isoprene during fermentation was 6.1%. The weightpercent yield of isoprene from glucose was 2.8%.

Example 10 Production of Isoprene in E. coli Expressing RecombinantKudzu Isoprene Synthase

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein E. coli:

The protein sequence for the kudzu (Pueraria montana) isoprene synthasegene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprenesynthase gene, optimized for E. coli codon usage, was purchased fromDNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from thesupplied plasmid by restriction endonuclease digestion withBspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen)that had been digested with NcoI/PstI. The construct was designed suchthat the stop codon in the isoprene synthase gene 5′ to the PstI site.As a result, when the construct was expressed the His-Tag is notattached to the isoprene synthase protein. The resulting plasmid,pTrcKudzu, was verified by sequencing (FIGS. 2 and 3).

The isoprene synthase gene was also cloned into pET16b (Novagen). Inthis case, the isoprene synthase gene was inserted into pET16b such thatthe recombinant isoprene synthase protein contained the N-terminal Histag. The isoprene synthase gene was amplified from pTrcKudzu by PCRusing the primer set pET-His-Kudzu-2F:5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) andpET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:4). These primers added an NdeI site at the 5′-end and a BamH1 siteat the 3′ end of the gene respectively. The plasmid pTrcKudzu, describedabove, was used as template DNA, Herculase polymerase (Stratagene) wasused according to manufacture's directions, and primers were added at aconcentration of 10 pMols. The PCR was carried out in a total volume of25 μl. The PCR product was digested with NdeI/BamH1 and cloned intopET16b digested with the same enzymes. The ligation mix was transformedinto E. coli Top10 (Invitrogen) and the correct clone selected bysequencing. The resulting plasmid, in which the kudzu isoprene synthasegene was expressed from the T7 promoter, was designated pETNHisKudzu(FIGS. 4 and 5).

The kudzu isoprene synthase gene was also cloned into the low copynumber plasmid pCL1920. Primers were used to amplify the kudzu isoprenesynthase gene from pTrcKudzu described above. The forward primer added aHindIII site and an E. coli consensus RBS to the 5′ end. The PstIcloning site was already present in pTrcKudzu just 3′ of the stop codonso the reverse primer was constructed such that the final PCR productincludes the PstI site. The sequences of the primers were:HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQID NO:6) and BamH1-Kudzu R:

(SEQ ID NO: 4) 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG.

The PCR product was amplified using Herculase polymerase with primers ata concentration of 10 pmol and with 1 ng of template DNA (pTrcKudzu).The amplification protocol included 30 cycles of (95° C. for 1 minute,60° C. for 1 minute, 72° C. for 2 minutes). The product was digestedwith HindIII and PstI and ligated into pCL1920 which had also beendigested with HindIII and PstI. The ligation mix was transformed into E.coli Top10. Several transformants were checked by sequencing. Theresulting plasmid was designated pCL-lac-Kudzu (FIGS. 6 and 7A-7C).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred fromshake flasks to 20 ml CTC headspace vials (Agilent vial cat #5188 2753;cap cat #5188 2759). The cap was screwed on tightly and the vialsincubated at the equivalent temperature with shaking at 250 rpm. After30 minutes the vials were removed from the incubator and analyzed asdescribed below (see Table 1 for some experimental values from thisassay).

In cases where isoprene production in fermentors was determined, sampleswere taken from the off-gas of the fermentor and analyzed directly asdescribed below (see Table 2 for some experimental values from thisassay).

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The sampler was setup to inject 500 μL of headspace gas. The GC/MS method utilized heliumas the carrier gas at a flow of 1 ml/min. The injection port was held at250° C. with a split ratio of 50:1. The oven temperature was held at 37°C. for the 2 minute duration of the analysis. The Agilent 5793N massselective detector was run in single ion monitoring (SIM) mode on m/z67. The detector was switched off from 1.4 to 1.7 minutes to allow theelution of permanent gases. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. Acalibration table was used to quantify the absolute amount of isopreneand was found to be linear from 1 μg/L to 2000 μg/L. The limit ofdetection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. coli CellsExpressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21(Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu andBL21/pETHisKudzu. The strains were spread for isolation onto LA (Luriaagar)+carbenicillin (50 μg/ml) and incubated overnight at 37° C. Singlecolonies were inoculated into 250 ml baffled shake flasks containing 20ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml). Cultures weregrown overnight at 20° C. with shaking at 200 rpm. The OD₆₀₀ of theovernight cultures were measured and the cultures were diluted into a250 ml baffled shake flask containing 30 ml MagicMedia(Invitrogen)+carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culture wasincubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8, 400μM IPTG was added and the cells were incubated for a further 6 hours at30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours after inductionwith IPTG, 1 ml aliquots of the cultures were collected, the OD₆₀₀ wasdetermined and the amount of isoprene produced was measured as describedabove. Results are shown in FIGS. 8A-8D.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing therecombinant kudzu isoprene synthase gene was determined from a fed-batchculture. The recipe for the fermentation media (TM2) per liter offermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,MgSO4*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) andq.s. to volume. The final product was filter sterilized with 0.22μfilter (only, do not autoclave). The recipe for 1000× Modified TraceMetal Solution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g,NaCl 10 g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O100 mg, H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolvedone a time in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume andfilter sterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isopreneformation from glucose at the desired fermentation, pH 6.7 andtemperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu takenfrom a frozen vial was prepared in soytone-yeast extract-glucose medium.After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifugedand the contents resuspended in 70 ml supernatant to transfer the cellpellet (70 ml of OD 3.1 material) to the bioreactor. At various timesafter inoculation, samples were removed and the amount of isopreneproduced was determined as described above. Results are shown in FIGS.9A and 9B.

Example 11 Production of Isoprene in E. coli Expressing RecombinantPoplar Isoprene Synthase

The protein sequence for the poplar (Populus alba×Populus tremula)isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786)was obtained from GenBank (CAC35696). A gene, codon optimized for E.coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31A and31B). The isoprene synthase gene was removed from the supplied plasmidby restriction endonuclease digestion with BspLU11I/PstI, gel-purified,and ligated into pTrcHis2B that had been digested with NcoI/PstI. Theconstruct is cloned such that the stop codon in the insert is before thePstI site, which results in a construct in which the His-Tag is notattached to the isoprene synthase protein. The resulting plasmidpTrcPoplar (FIGS. 32 and 33A-33C), was verified by sequencing.

Example 12 Production of Isoprene in Panteoa citrea ExpressingRecombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 10 wereelectroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformantswere selected on LA containing carbenicillin (200 μg/ml) orspectinomycin (50 μg/ml) respectively. Production of isoprene from shakeflasks and determination of the amount of isoprene produced wasperformed as described in Example 10 for E. coli strains expressingrecombinant kudzu isoprene synthase. Results are shown in FIGS. 10A-10C.

Example 13 Production of Isoprene in Bacillus subtilis ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of a B. subtilis Replicating Plasmid for the Expressionof Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilisaprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid(pBS19 with a chloramphenicol resistance cassette) under control of theaprE promoter. The isoprene synthase gene, the aprE promoter and thetranscription terminator were amplified separately and fused using PCR.The construct was then cloned into pBS19 and transformed into B.subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillussubtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 58)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-43 (−) Fuse aprE promoter to Kudzu ispS (SEQ ID NO: 59)5′-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTAb) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu(SEQ ID NO:2). The gene had been codon optimized for E. coli andsynthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the 3′end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCc) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillusamyliquefaciens was amplified from a previously sequenced plasmidpJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 62)5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGGCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR withthe following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAATCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu-terminator fragment was fused to the promoter fragment usingPCR with the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 64)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes MfeI and BamHI. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated to a vector known aspBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA+50 carbenicillin plates. A total of six colonieswere chosen and grown overnight in LB+50 carbenicillin and then plasmidswere isolated using a Qiagen kit. The plasmids were digested with EcoRIand BamHI to check for inserts and three of the correct plasmids weresent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter (SEQ ID NO: 65)5′-GACATGAATTCCTCCATTTTCTTCTGC CF 847 (+) Sequence in pXX 049 (end ofaprE promoter) (SEQ ID NO: 66) 5′-AGGAGAGGGTAAAGAGTGAG CF 07-45 (−) Fusethe 3′ end of kudzu isoprene synthase to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC CF 07-48 (+) Sequencingprimer for kudzu isoprene synthase (SEQ ID NO: 67)5′-CTTTTCCATCACCCACCTGAAG CF 07-49 (+) Sequencing in kudzu isoprenesynthase (SEQ ID NO: 68) 5′-GGCGAAATGGTCCAACAACAAAATTATC

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12A-12C) was correctby sequencing and was transformed into BG 3594 comK, a Bacillus subtilishost strain. Selection was done on LA+5 chloramphenicol plates. Atransformant was chosen and struck to single colonies on LA+5chloramphenicol, then grown in LB+5 chloramphenicol until it reached anOD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in the presenceof glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis Cellsexpressing Recombinant Isoprene Synthase

Overnight cultures were inoculated with a single colony of CF 443 from aLA+ Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB+Cm at 37°C. with shaking at 200 rpm. These overnight cultures (1 ml) were used toinoculate 250 ml baffled shake flasks containing 25 ml Grants II mediaand chloramphenicol at a final concentration of 25 μg/ml. Grants IIMedia recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 g urea,100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was 83.72 gMOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄ solution, 10ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100× micronutrients q.s.to 1 L with H₂O; and 100× micronutrients recipe was 1.47 g CaCl₂*2H₂O,0.4 g FeSO₄*7H₂0, 0.1 g MnSO₄*H₂O, 0.1 g ZnSO₄*H₂O, 0.05 g CuCl₂*2H₂O,0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄*2H₂O, q.s. to 1 L with H₂O. Shake flaskswere incubated at 37° C. and samples taken at 18, 24, and 44 hours. At18 hours the headspaces of CF443 and the control strain were sampled.This represented 18 hours of accumulation of isoprene. The amount ofisoprene was determined by gas chromatography as described in Example10. Production of isoprene was enhanced significantly by expressingrecombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing therecombinant kudzu isoprene synthase gene on a replication plasmid wasdetermined from a fed-batch culture. Bacillus strain CF 443, expressinga kudzu isoprene synthase gene, or control stain which does not expressa kudzu isoprene synthase gene were cultivated by conventional fed-batchfermentation in a nutrient medium containing soy meal (Cargill), sodiumand potassium phosphate, magnesium sulfate and a solution of citricacid, ferric chloride and manganese chloride. Prior to fermentation themedia is macerated for 90 minutes using a mixture of enzymes includingcellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batchfermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADMVersadex greens or Danisco invert sugar) and 99% wt/wt oil (WesternFamily soy oil, where the 99% wt/wt is the concentration of oil beforeit was added to the cell culture medium). Feed was started when glucosein the batch was non-detectable. The feed rate was ramped over severalhours and was adjusted to add oil on an equal carbon basis. The pH wascontrolled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case offoaming, antifoam agent was added to the media. The fermentationtemperature was controlled at 37° C. and the fermentation culture wasagitated at 750 rpm. Various other parameters such as pH, DO %, airflow,and pressure were monitored throughout the entire process. The DO % ismaintained above 20. Samples were taken over the time course of 36 hoursand analyzed for cell growth (OD₅₅₀) and isoprene production. Results ofthese experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid(pJH101-cmpR) under the control of the aprE promoter. Under theconditions tested, no isoprene was detected.

Example 14 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized byDNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served as the template forthe following PCR amplification reaction: 1 μl plasmid template (20ng/ul), 1 μl Primer EL-945 (10 uM)5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 μl PrimerEL-965 (10 uM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:10), 1 μldNTP (10 mM), 5 μl 10× PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μlPfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reactionvolume of 50 μl. The forward primer contained an additional 4nucleotides at the 5′-end that did not correspond to the Y. lipolyticacodon-optimized kudzu isoprene synthase gene, but was required forcloning into the pENTR/D-TOPO vector. The reverse primer contained anadditional 21 nucleotides at the 5′-end that did not correspond to theY. lipolytica codon-optimized kudzu isoprene synthase gene, but wereinserted for cloning into other vector backbones. Using the MJ ResearchPTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C.for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1minute after the last cycle. The PCR product was analyzed on a 1.2%E-gel to confirm successful amplification of the Y. lipolyticacodon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kitfollowing manufacturer's protocol: 1 μl PCR reaction, 1 μl Saltsolution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a totalreaction volume of 6 μl. The reaction was incubated at room temperaturefor 5 minutes. One microliter of TOPO reaction was transformed intoTOP10 chemically competent E. coli cells. The transformants wereselected on LA+50 μg/ml kanamycin plates. Several colonies were pickedand each was inoculated into a 5 ml tube containing LB+50 μg/mlkanamycinand the cultures grown overnight at 37° C. with shaking at 200 rpm.Plasmids were isolated from the overnight culture tubes using QIAprepSpin Miniprep Kit, following manufacturer's protocol. Several plasmidswere sequenced to verify that the DNA sequence was correct.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimizedkudzu isoprene synthase gene, was used for Gateway Cloning into acustom-made pTrex3g vector. Construction of pTrex3g is described in WO2005/001036 A2. The reaction was performed following manufacturer'sprotocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μlY. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPOdonor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 ina total reaction volume of 8 μl. The reaction was incubated at roomtemperature for 1 hour and then 1 μl proteinase K solution was added andthe incubation continued at 37° C. for 10 minutes. Then 1 μl of reactionwas transformed into TOP10 chemically competent E. coli cells. Thetransformants were selected on LA+50 μg/ml carbenicillin plates. Severalcolonies were picked and each was inoculated into a 5 ml tube containingLB+50 μg/ml carbenicillin and the cultures were grown overnight at 37°C. with shaking at 200 rpm. Plasmids were isolated from the overnightculture tubes busing QIAprep Spin Miniprep Kit (Qiagen, Inc.), followingmanufacturer's protocol. Several plasmids were sequenced to verify thatthe DNA sequence was correct.

Biolistic transformation of Y. lipolytica codon-optimized kudzu isoprenesynthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reeseistrain was performed using the Biolistic PDS-1000/HE Particle DeliverySystem (see WO 2005/001036 A2). Isolation of stable transformants andshake flask evaluation was performed using protocol listed in Example 11of patent publication WO 2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above were transferred to head space vials. The vials weresealed and incubated for 5 hours at 30° C. Head space gas was measuredand isoprene was identified by the method described in Example 10. Twoof the transformants showed traces of isoprene. The amount of isoprenecould be increased by a 14 hour incubation. The two positive samplesshowed isoprene at levels of about 0.5 μg/L for the 14 hour incubation.The untransformed control showed no detectable levels of isoprene. Thisexperiment shows that T. reesei is capable of producing isoprene fromendogenous precursor when supplied with an exogenous isoprene synthase.

Example 15 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Yarrowia lipolytica.

The starting point for the construction of vectors for the expression ofthe kudzu isoprene synthase gene in Yarrowia lipolytica was the vectorpSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID No:11) isshown in FIGS. 15A-15C.

The following fragments were amplified by PCR using chromosomal DNA of aY. lipolytica strain GICC 120285 as the template: a promotorless form ofthe URA3 gene, a fragment of 18S ribosomal RNA gene, a transcriptionterminator of the Y. lipolytica XPR2 gene and two DNA fragmentscontaining the promoters of XPR2 and ICL1 genes. The following PCRprimers were used:

ICL1 3 5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGGTGAC (SEQ IDNO: 69) ICL1 5 5′-GCAGGTGGGAAACTATGCACTCC (SEQ ID NO: 70) XPR 35′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG (SEQ ID NO: 71) XPR 55′-GGTGTCGACGTACGGTCGAGCTTATTGACC (SEQ ID NO: 72) XPRT35′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG (SEQ ID NO: 73) XPRT 55′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG (SEQ ID NO: 74) Y18S35′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG (SEQ ID NO: 75) Y18S 55′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG (SEQ ID NO: 76) YURA35′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG (SEQ ID NO: 77) YURA 505′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG (SEQ ID NO: 78) YURA 515′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC (SEQ ID NO: 79)

For PCR amplification the PfuUltraII polymerase (Stratagene),supplier-provided buffer and dNTPs, 2.5 μM primers and the indicatedtemplate DNA were used as per the manufacturer's instructions. Theamplification was done using the following cycle: 95° C. for 1 min;34×(95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3 min) and 10 minat 72° C. followed by a 4° C. incubation

Synthetic DNA molecules encoding the kudzu isoprene synthase gene,codon-optimized for expression in Yarrowia, was obtained from DNA 2.0(FIG. 16; SEQ ID NO:12). Full detail of the construction scheme of theplasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprenesynthase gene under control of XPR2 and ICL1 promoters respectively ispresented in FIGS. 18A-18F. Control plasmids in which a mating factorgene (MAP29) is inserted in place of an isoprene synthase gene were alsoconstructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populusalba×Populus tremula) isoprene synthase gene. The sequence of the poplarisoprene is described in Miller B. et al. (2001) Planta 213, 483-487 andshown in FIG. 17 (SEQ ID NO:13). A construction scheme for thegeneration the plasmids pYLA(POP1) and pYLI(POP1) carrying syntheticpoplar isoprene synthase gene under control of XPR2 and ICL1 promotersrespectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica.

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digestedwith SacII and used to transform the strain Y. lipolytica CLIB 122 by astandard lithium acetate/polyethylene glycol procedure to uridineprototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract,2% peptone, 2% glucose) overnight, were collected by centrifugation(4000 rpm, 10 min), washed once with sterile water and suspended in 0.1M lithium acetate, pH 6.0. Two hundred μl aliquots of the cellsuspension were mixed with linearized plasmid DNA solution (10-20 μg),incubated for 10 minutes at room temperature and mixed with 1 ml of 50%PEG 4000 in the same buffer. The suspensions were further incubated for1 hour at room temperature followed by a 2 minutes heat shock at 42° C.Cells were then plated on SC his leu plates (0.67% yeast nitrogen base,2% glucose, 100 mg/L each of leucine and histidine). Transformantsappeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates fromthe pYLI(KZ1) transformation, two isolates from the pYLA(MAP29)transformation and two isolates from the pYLI(MAP29) transformation weregrown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0)at 30° C. with shaking. Cells from 10 ml of culture were collected bycentrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 mlscrew cap vials. The vials were incubated overnight at room temperaturewith gentle (60 rpm) shaking. Isoprene content in the headspace of thesevials was analyzed by gas chromatography using mass-spectrometricdetector as described in Example 10. All transformants obtained withpYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene(0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspaceof the control strains carrying phytase gene instead of an isoprenesynthase gene.

Example 16 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, ordxs, or idi and dxs for the Production of Isoprene in E. coli

i) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 10) was replaced withthe gene conferring kanamycin resistance. To remove the bla gene,pTrcKudzu was digested with BspHI, treated with Shrimp AlkalinePhosphatase (SAP), heat killed at 65° C., then end-filled with Klenowfragment and dNTPs. The 5 kbp large fragment was purified from anagarose gel and ligated to the kan^(r) gene which had been PCR amplifiedfrom pCR-Blunt-II-TOPO using primers MCM225′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) and MCM235′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested withHindIII and PvuI, and end-filled. A transformant carrying a plasmidconferring kanamycin resistance (pTrcKudzuKan) was selected on LAcontaining kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed andgel purified. It was ligated to a PCR product encoding idi from S.cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI 1R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S.cerevisiae genomic DNA. The PCR product was digested with NsiI and PstIand gel purified prior to ligation. The ligation mixture was transformedinto chemically competent TOP10 cells and selected on LA containing 50μg/ml kanamycin. Several transformants were isolated and sequenced andthe resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and35A-35C).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding dxsfrom E. coli with a synthetic RBS. The primers for PCR were MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); andthe template was E. coli genomic DNA. The PCR product was digested withNsiI and PstI and gel purified prior to ligation. The resultingtransformation reaction was transformed into TOP10 cells and selected onLA with kanamycin 50 μg/ml. Several transformants were isolated andsequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS.36 and 37A-37C).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding E.coli dxs with a synthetic RBS (primers MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCC G (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19);template TOP10 cells) which had been digested with NsiI and PstI and gelpurified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS.21 and 22A-22D).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene andterminator from Example 10 above was digested from pTrcKudzu using SspIand gel purified. It was ligated to pCL1920 which had been digested withPvuII, treated with SAP and heat killed. The resulting ligation mixturewas transformed into TOP10 cells and selected in LA containingspectinomycin 50 μg/ml. Several clones were isolated and sequenced andtwo were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insertin opposite orientations (FIGS. 38-41A-41C).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) abovewas ligated into pCL PtrcKudzu which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu yIDI (FIGS. 42 and 43A-43C).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) abovewas ligated into pCL PtrcKudzu (A3) which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu DXS (FIGS. 44 and 45A-45D).

II. Measurement of Isoprene in Headspace from Cultures Expressing KudzuIsoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λDE3) previously transformed with plasmidspTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C),pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Culturesof pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCLPtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL.

Cultures were induced with 400 μM IPTG at time 0 (OD₆₀₀ approximately0.5) and samples taken for isoprene headspace measurement (see Example10). Results are shown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS wasgrown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and usedto inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 gMgSO₄*7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammoniumcitrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× ModifiedTrace Metal Solution, adjusted to pH 6.8 and q.s. to H₂0, and filtersterilized) containing 1% glucose. Flasks were incubated at 30° C. untilan OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Sampleswere taken at various times after induction and the amount of isoprenein the head space was measured as described in Example 10. Results areshown in FIG. 23H.

III. The Effect of Yeast Extract on Isoprene Production in E. coli Grownin Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously describedwith E. coli cells containing the pTrcKudzu yIDI DXS plasmid describedabove. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed atan exponential rate. The total amount of yeast extract delivered to thefermentor was varied between 70-830 g during the 40 hour fermentation.Optical density of the fermentation broth was measured at a wavelengthof 550 nm. The final optical density within the fermentors wasproportional to the amount of yeast extract added (FIG. 48A). Theisoprene level in the off-gas from the fermentor was determined aspreviously described. The isoprene titer increased over the course ofthe fermentation (FIG. 48B). The amount of isoprene produced waslinearly proportional to the amount of fed yeast extract (FIG. 48C).

IV. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21(XDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels ofisoprene varied from 50 to 300 μg/L over a time period of 15 hours. Onthe basis of the average isoprene concentrations, the average flowthrough the device and the extent of isoprene breakthrough, the amountof isoprene collected was calculated to be approximately 17 g.

V. Production of Isoprene in 500 L Fermentation of E. coli Grown inFed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g,and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cellscontaining the pTrcKudzu yIDI DXS plasmid. This experiment was carriedout to monitor isoprene formation from glucose and yeast extract at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was prepared in soytone-yeastextract-glucose medium. After the inoculum grew to OD 0.15, measured at550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-Lsoytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose werefed at exponential rates. The total amount of glucose and yeast extractdelivered to the bioreactor during the 50 hour fermentation was 181.2 kgand 17.6 kg, respectively. The optical density within the bioreactorover time is shown in FIG. 49A. The isoprene level in the off-gas fromthe bioreactor was determined as previously described. The isoprenetiter increased over the course of the fermentation (FIG. 49B). Thetotal amount of isoprene produced during the 50 hour fermentation was55.1 g and the time course of production is shown in FIG. 49C.

Example 17 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and Recombinant Mevalonic Acid Pathway Genes

I. Cloning the Lower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows.Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase(MVD) and isopentenyl diphosphate isomerase genes were amplified by PCRfrom S. cerevisiae chromosomal DNA and cloned individually into the pCRBluntII TOPO plasmid (Invitrogen). In some cases, the idi gene wasamplified from E. coli chromosomal DNA. The primers were designed suchthat an E. coli consensus RBS (AGGAGGT (SEQ ID NO:80) or AAGGAGG (SEQ IDNO:81)) was inserted at the 5′ end, 8 bp upstream of the start codon anda PstI site was added at the 3′ end. The genes were then cloned one byone into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC204508D). The MVK gene was amplified from the chromosome of S.cerevisiae using primers MVKF(5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:21) andMVK-PstI-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQID NO:22) using PfuTurbo as per manufacturer's instructions. The correctsized PCR product (1370 bp) was identified by electrophoresis through a1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resultingplasmid was designated pMVK1. The plasmid pMVK1 was digested with SacIand TaqI restriction endonucleases and the fragment was gel purified andligated into pTrcHis2B digested with SacI and BstBI. The resultingplasmid was named pTrcMVK1 (also referred to as pTrcK).

The second gene in the mevalonic acid biosynthesis pathway, PMK, wasamplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC,SEQ ID NO:23) and BsiHKA I-PMK1 F(5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:24). The PCRreaction was performed using Pfu Turbo polymerase (Stratagene) as permanufacturer's instructions. The correct sized product (1387 bp) wasdigested with PstI and BsiHKI and ligated into pTrcMVK1 digested withPstI. The resulting plasmid was named pTrcKK.

The MVD and the idi genes were cloned in the same manner. PCR wascarried out using the primer pairs PstI-MVD 1 R(5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:25) and NsiI-MVD 1 F(5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:26) to amplify the MVDgene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC, SEQ ID NO:27) andNsiI-YIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:28)to amplify the yIDI gene. The plasmid with the MVK, PMK, and MVD genesinserted is named pTrcKKD. In some cases the IPP isomerase gene, idifrom E. coli was used. To amplify idi from E. coli chromosomal DNA, thefollowing primer set was used: PstI-CIDI 1 R(5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:29) and NsiI-CIDI 1 F(5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ ID NO:30). Template DNAwas chromosomal DNA isolated by standard methods from E. coli FM5 (WO96/35796 and WO 2004/033646, which are each hereby incorporated byreference in their entireties, particularly with respect to isolation ofnucleic acids). The final plasmids were named pKKDIy for the constructencoding the yeast idi gene or pKKDIc for the construct encoding the E.coli idi gene. The plasmids were transformed into E. coli hosts BL21 forsubsequent analysis. In some cases the isoprene synthase from kudzu wascloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycinantibiotic resistance marker. The plasmid pTrcKKDIy was digested withrestriction endonucleases ApaI and PstI, the 5930 bp fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. Theplasmid pTrcKudzuKan, described in Example 16, was digested withrestriction endonucleases ApaI and PstI, and the 3338 bp fragmentcontaining the vector was purified from a 1.2% E-gel using the QiagenGel Purification kit. The 3338 bp vector fragment and the 5930 bp lowerMVA pathway fragment were ligated using the Roche Quick Ligation kit.The ligation mix was transformed into E. coli TOP10 cells andtranformants were grown at 37° C. overnight with selection on LAcontaining kanamycin (50 μg/ml). The transformants were verified byrestriction enzyme digestion and one was frozen as a stock. The plasmidwas designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu,described in Example 10, using primers MCM505′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAATTTAC T (SEQ IDNO:31) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:32). The resulting PCR fragment was cloned into pCR2.1 andtransformed into E. coli TOP10. This fragment contains the codingsequence for kudzu isoprene synthase and an upstream region containing aRBS from E. coli. Transformants were incubated overnight at 37° C. withselection on LA containing carbenicillin (50 μg/ml). The correctinsertion of the fragment was verified by sequencing and this strain wasdesignated MCM93.

The plasmid from strain MCM93 was digested with restrictionendonucleases NsiI and PstI to liberate a 1724 bp insert containing theRBS and kudzu isoprene synthase. The 1724 bp fragment was separated on a1.2% agarose E-gel and purified using the Qiagen Gel Purification kitaccording to the manufacturer's instructions. Plasmid pTrcKanKKDIy wasdigested with the restriction endonuclease PstI, treated with SAP for 30minutes at 37° C. and purified using the Qiagen PCR cleanup kit. Theplasmid and kudzu isoprene synthase encoding DNA fragment were ligatedusing the Roche Quick Ligation kit. The ligation mix was transformedinto E. coli TOP10 cells and transformants were grown overnight at 37°C. with selection on LA containing Kanamycin at 50 μg/ml. The correcttransformant was verified by restriction digestion and the plasmid wasdesignated pTrcKKDyIkISKan (FIGS. 24 and 25A-25D). This plasmid wastransformed into BL21(λDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing theRecombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt etal., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 andsupplemented with 0.5% glucose and 0.5% mevalonic acid. A controlculture was also set up using identical conditions but without theaddition of 0.5% mevalonic acid. The culture was started from anovernight seed culture with a 1% inoculum and induced with 500 μM IPTGwhen the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures weregrown at 30° C. with shaking at 250 rpm. The production of isoprene wasanalyzed 3 hours after induction by using the head space assay describedin Example 10. Maximum production of isoprene was 6.67×10⁻⁴mol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth andincludes both the volume of the cell medium and the volume of the cells.The control culture not supplemented with mevalonic acid did not producemeasurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encodingthree enzymatic activities, was cloned from Enterococcus faecalis. ThemvaE gene encodes a protein with the enzymatic activities of bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, the first and third proteins in the pathway, andthe mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase.The mvaE gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with an E. coli ribosome binding site and a spacer in frontusing the following primers:

CF 07-60 (+) Start of mvaE w/RBS + ATG start codon SacI5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTATTG (SEQ ID NO: 34) CF07-62 (−) Fuse mvaE to mvaS with RBS in between5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATC (SEQ ID NO:35)

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with a RBS and spacer from E. coli in front using thefollowing primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAA (SEQ ID NO:36) CF 07-102 (−) End of mvaS gene BglII5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT (SEQ ID NO: 37)

The PCR fragments were fused together with PCR using the followingprimers:

CF 07-60 (+) Start of mvaE w/RBS + ATG start codon SacI5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTATTATTG (SEQ ID NO: 34) CF07-102 (−) End of mvaS gene BglII5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT (SEQ ID NO: 37)

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes SacI and BglII. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated into the commerciallyavailable vector pTrcHis2A, which had been digested with SacI and BglIIand gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA+50 μg/ml carbenicillin plates. A total of sixcolonies were chosen and grown overnight in LB+50 μg/ml carbenicillinand plasmids were isolated using a Qiagen kit. The plasmids weredigested with SacI and BglII to check for inserts and one correctplasmid was sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene (SEQ ID NO:39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start of mvaS gene(SEQ ID NO: 40) 5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaSgene (SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+) Sequencein mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+) Sequencein mvaE (SEQ ID NO: 43) 5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+)Sequence in mvaE (SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89(+) Sequence mvaS (SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing andwas transformed into the commercially available E. coli strain BL21.Selection was done on LA+50 μg/ml carbenicillin. Two transformants werechosen and grown in LB+50 μg/ml carbenicillin until they reached anOD₆₀₀ of 1.5. Both strains were frozen in a vial at −80° C. in thepresence of glycerol. Strains were designated CF 449 forpTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 forpTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found tobehave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restrictionendonuclease SspI to release a fragment containing pTrc-mvaE-mvaS-(Histag)-terminator. In this fragment, the his-tag was not translated. Thisblunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using theQiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbpfragment from pCL1920 was prepared by digesting the vector with therestriction endonuclease PvuII, treating with SAP and gel purifying froma 1.2% E-gel using the Qiagen Gel Purification kit. The two fragmentswere ligated using the Roche Quick Ligation Kit and transformed intoTOP10 chemically competent cells. Transformants were selected on LAcontaining spectinomycin (50 μg/ml). A correct colony was identified byscreening for the presence of the insert by PCR. The plasmid wasdesignated pCL PtrcUpperPathway (FIGS. 26 and 27A-27D).

VI. Strains Expressing the Combined Upper and Lower Mevalonic AcidPathways

To obtain a strain with a complete mevalonic acid pathway plus kudzuisoprene synthase, plasmids pTrcKKDyIkISkan and pCLpTrcUpperPathway wereboth transformed into BL21(λDE3) competent cells (Invitrogen) andtransformants were selected on LA containing kanamycin (50 μg/ml) andSpectinomycin (50 μg/ml). The transformants were checked by plasmid prepto ensure that both plasmids were retained in the host. The strain wasdesignated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/ppTrcHis2A-mvaE/mvaS are inoculated into LB+carbenicillin (100 μg/ml) andare grown overnight at 37° C. with shaking at 200 rpm. These cultureswere diluted into 50 ml medium in 250 ml baffled flasks to an OD₆₀₀ of0.1. The medium was TM3+1 or 2% glucose+carbenicillin (100 ug/ml) orTM3+1% glucose+hydrolyzed soy oil+carbenicillin (100 ug/ml) orTM3+biomass (prepared bagasse, corn stover or switchgrass). Cultureswere grown at 30° C. with shaking at 200 rpm for approximately 2-3 hoursuntil an OD₆₀₀ of 0.4 was reached. At this point the expression from themvaE mvaS construct was induced by the addition of IPTG (400 μM).Cultures were incubated for a further 20 or 40 hours with samples takenat 2 hour intervals to 6 hour post induction and then at 24, 36 and 48hours as needed. Sampling was done by removing 1 ml of culture,measuring the OD₆₀₀, pelleting the cells in a microfuge, removing thesupernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid with TM3medium and 2% glucose as the cell medium. A shake flask of these cellsproduced 2-4 grams of mevalonic acid per liter with LB medium and 1%glucose as the cell culture medium. The production of mevalonic acid inthese strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. coli BL21 Containing the Upper andLower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in variouscombinations of plasmids containing the upper and lower MVA pathway andthe kudzu isoprene synthase gene as described above and the plasmidscontaining the idi, dxs, and dxr and isoprene synthase genes describedin Example 16. The host cells used were chemically competent BL21(λDE3)and the transformations were done by standard methods. Transformantswere selected on L agar containing kanamycin (50 μg/ml) or kanamycinplus spectinomycin (both at a concentration of 50 μg/ml). Plates weregrown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin plus Spectinomycin (50 μg/ml each)

-   MCM127—pCL Upper MVA+pTrcKKDyIkIS (kan) in BL21(λDE3)-   MCM131—pCL1920+pTrcKKDyIkIS (kan) in BL21(λDE3)-   MCM125—pCL Upper MVA+pTrcHis2B (kan) in BL21(λDE3)

Grown on Kanamycin (50 μg/ml)

-   MCM64—pTrcKudzu yIDI DXS (kan) in BL21(λDE3)-   MCM50—pTrcKudzu (kan) in BL21(λDE3)-   MCM123—pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA+appropriateantibiotic and grown overnight at 37° C. A single colony from each platewas used to inoculate shake flasks (25 ml LB+the appropriateantibiotic). The flasks were incubated at 22° C. overnight with shakingat 200 rpm. The next morning the flasks were transferred to a 37° C.incubator and grown for a further 4.5 hours with shaking at 200 rpm. The25 ml cultures were centrifuged to pellet the cells and the cells wereresuspended in 5 ml LB+the appropriate antibiotic. The cultures werethen diluted into 25 ml LB+1% glucose+the appropriate antibiotic to anOD₆₀₀ of 0.1. Two flasks for each strain were set up, one set forinduction with IPTG (800 μM) the second set was not induced. Thecultures were incubated at 37° C. with shaking at 250 rpm. One set ofthe cultures were induced after 1.50 hours (immediately followingsampling time point 1). At each sampling time point, the OD₆₀₀ wasmeasured and the amount of isoprene determined as described in Example10. Results are presented in Table 10. The amount of isoprene made ispresented as the amount at the peak production for the particularstrain.

TABLE 10 Production of isoprene in E. coli strains Strain Isoprene(μg/liter/OD/hr MCM50 23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127 874ND: not detectedTrace: peak present but not integrable.IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS #503-48-0) was supplied fromSigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL)and was treated with potassium hydroxide (7.7 mmol) in order to generatethe potassium salt of mevalonic acid. The conversion to mevalonic acidwas confirmed by ¹H NMR analysis. Samples for HPLC analysis wereprepared by centrifugation at 14,000 rpm for 5 minutes to remove cells,followed by the addition of a 300 μl aliquot of supernatant to 900 μl ofH₂O. Perchloric acid (36 μl of a 70% solution) was then added followedby mixing and cooling on ice for 5 minutes. The samples were thencentrifuged again (14,000 rpm for 5 min) and the supernatant transferredto HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) wereprepared in the same fashion. Analysis of mevalonic acid (20 uLinjection volume) was performed by HPLC using a BioRad Aminex87-H+column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6mL/min with refractive index (RI) detection. Under these conditionsmevalonic acid eluted as the lactone form at 18.5 minutes.

X. Production of Isoprene from E. coli BL21 Containing the Upper MVAPathway Plus Kudzu Isoprene Synthase

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 2.2 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperPathway (FIG. 26) and pTrcKKDyIkISplasmids. This experiment was carried out to monitor isoprene formationfrom glucose at the desired fermentation pH 7.0 and temperature 30° C.An inoculum of E. coli strain taken from a frozen vial was streaked ontoan LB broth agar plate (with antibiotics) and incubated at 37° C. Asingle colony was inoculated into soytone-yeast extract-glucose medium.After the inoculum grew to OD 1.0 when measured at 550 nm, 500 mL wasused to inoculate a 15-L bioreactor containing an initial working volumeof 5 L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 3.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 uM when the optical density at 550nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to50 uM when OD₅₅₀ reached 190. IPTG concentration was raised to 100 uM at38 hours of fermentation. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 54. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titerincreased over the course of the fermentation to a final value of 2.2g/L (FIG. 55). The total amount of isoprene produced during the 54 hourfermentation was 15.9 g, and the time course of production is shown inFIG. 56.

XI. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides and Kudzu isoprene synthase was used to produce isoprenefrom cells in fed-batch culture. This experiment demonstrates thatgrowing cells under glucose limiting conditions resulted in theproduction of 3.0 g/L of isoprene.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× Modified Trace Metal Solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-Lbioreactor containing an initial working volume of 5 L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 59 hour fermentation was 2.2 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 93. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.0 g/L (FIG. 94). The total amount ofisoprene produced during the 59 hour fermentation was 22.8 g, and thetime course of production is shown in FIG. 95. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.2%. The weight percent yield of isoprene from glucose was 1.0%.

XII. Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale

A 15-L scale fermentation of E. coli expressing mevalonic acid pathwaypolypeptides, Pueraria lobata isoprene synthase, and Kudzu isoprenesynthase was used to produce isoprene from cells in fed-batch culture.This experiment demonstrates that growing cells under glucose limitingconditions resulted in the production of 3.3 g/L of isoprene.

i) Construction of pCLPtrcUpperPathwayHGS2

The gene encoding isoprene synthase from Pueraria lobata wasPCR-amplified using primers NsiI-RBS-HGS F(CTTGATGCATCCTGCATTCGCCCTTAGGAGG, SEQ ID NO:88) and pTrcR(CCAGGCAAATTCTGTTTTATCAG, SEQ ID NO:89), and pTrcKKDyIkIS as a template.The PCR product thus obtained was restriction-digested with NsiI andPstI and gel-purified. The plasmid pCL PtrcUpperPathway wasrestriction-digested with PstI and dephosphorylated using rAPid alkalinephosphatase (Roche) according to manufacturer's instructions.

These DNA fragments were ligated together and the ligation reaction wastransformed into E. coli Top10 chemically competent cells (Invitrogen),plated on L agar containing spectinomycin (50 ug/ml) and incubatedovernight at 37° C. Plasmid DNA was prepared from 6 clones using theQiaquick Spin Mini-prep kit. The plasmid DNA was digested withrestriction enzymes EcoRV and MluI to identify a clone in which theinsert had the right orientation (i.e., the gene oriented in the sameway as the pTrc promoter).

The resulting correct plasmid was designated pCLPtrcUpperPathwayHGS2.This plasmid was assayed using the headspace assay described herein andfound to produce isoprene in E. coli Top10, thus validating thefunctionality of the gene. The plasmid was transformed into BL21(LDE3)containing pTrcKKDyIkIS to yield the strainBL21/pCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS. This strain has an extra copyof the isoprene synthase compared to the BL21/pCL PtrcUpperMVA and pTrcKKDyIkIS strain (Example 17, part XI). This strain also had increasedexpression and activity of HMGS compared to the BL21/pCL PtrcUpperMVAand pTrc KKDyIkIS strain used in Example 17, part XI.

ii) Isoprene Fermentation from E. coli ExpressingpCLPtrcUpperPathwayHGS2-pTrcKKDyIkIS and Grown in Fed-Batch Culture atthe 15-L Scale

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the pCLPtrcUpperPathwayHGS2 and pTrc KKDyIkIS plasmids.This experiment was carried out to monitor isoprene formation fromglucose at the desired fermentation pH 7.0 and temperature 30° C. Aninoculum of E. coli strain taken from a frozen vial was streaked onto anLB broth agar plate (with antibiotics) and incubated at 37° C. A singlecolony was inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0 measured at 550 nm, 500 mL was used to inoculatea 15-L bioreactor containing an initial working volume of 5 L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 58 hour fermentation was 2.1 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 9. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 170. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 104. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 3.3 g/L (FIG. 105). The total amount ofisoprene produced during the 58 hour fermentation was 24.5 g and thetime course of production is shown in FIG. 106. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 2.5%. The weight percent yield of isoprene from glucose was 1.2%.Analysis showed that the activity of the isoprene synthase was increasedby approximately 3-4 times that compared to BL21 expressing CLPtrcUpperMVA and pTrc KKDyIkIS plasmids (data not shown).

XIII. Chromosomal Integration of the Lower Mevalonate Pathway in E.coli.

A synthetic operon containing mevalonate kinase, mevalonate phosphatekinase, mevalonate pyrophosphate decarboxylase, and the IPP isomerasewas integerated into the chromosome of E. coli. If desired, expressionmay be altered by integrating different promoters 5′ of the operon.

Table 11 lists primers used for this experiment.

TABLE 11 Primers MCM78 attTn7 up rev for gcatgctcgagcggccgcTTTTintegration construct AATCAAACATCCTGCCAACTC (SEQ ID NO: 91) MCM79 attTn7down rev for gatcgaagggcgatcgTGTCAC integration constructAGTCTGGCGAAACCG (SEQ ID NO: 92) MCM88 attTn7 up forw forctgaattctgcagatatcTGTT integration construct TTTCCACTCTTCGTTCACTTT (SEQID NO: 93) MCM89 attTn7 down forw for tctagagggcccAAGAAAAATG integrationconstruct CCCCGCTTACG (SEQ ID NO: 94) MCM104 GI1.2 promoter-MVKGatcgcggccgcgcccttgacg atgccacatcctgagcaaataa ttcaaccactaattgtgagcggataacacaaggaggaaacagct atgtcattaccgttcttaactt c (SEQ ID NO: 95) MCM105aspA terminator-yIDI Gatcgggccccaagaaaaaagg cacgtcatctgacgtgccttttttatttgtagacgcgttgttat agcattcta (SEQ ID NO: 96) MCM120 Forward ofattTn7: aaagtagccgaagatgacggtt attTn7 homology, GBtgtcacatggagttggcaggat marker homology gtttgattaaaagcAATTAACCCTCACTAAAGGGCGG (SEQ ID NO: 97) MCM127 Rev complement of 1.2AGAGTGTTCACCAAAAATAATA GI: GB marker ACCTTTCCCGGTGCAgaagttahomology(extra long), agaacggtaatgacatagctgt promoter, RBS, ATGttcctccttgtgttatccgctc acaattagtggttgaattattt gctcaggatgtggcatcgtcaagggcTAATACGACTCACTATAG GGCTCG (SEQ ID NO: 98)i) Target Vector Construction

The attTn7 site was selected for integration. Regions of homologyupstream (attTn7 up) (primers MCM78 and MCM79) and downstream (attTn7down) (primers MCM88 and MCM89) were amplified by PCR from MG1655 cells.A 50 uL reaction with 1 uL 10 uM primers, 3 uL ddH2O, 45 uL InvitrogenPlatinum PCR Supermix High Fidelity, and a scraped colony of MG1655 wasdenatured for 2:00 at 94° C., cycled 25 times (2:00 at 94° C., 0:30 at50° C., and 1:00 at 68° C.), extended for 7:00 at 72° C., and cooled to4° C. This resulting DNA was cloned into pCR2.1 (Invitrogen) accordingto the manufacturer's instructions, resulting in plasmids MCM278 (attTn7up) and MCM252 (attTn7 down). The 832 bp ApaI-PvuI fragment digested andgel purified from MCM252 was cloned into ApaI-PvuI digested and gelpurified plasmid pR6K, creating plasmid MCM276. The 825 bp PstI-NotIfragment digested and gel purified from MCM278 was cloned into PstI-NotIdigested and gel purified MCM276, creating plasmid MCM281.

ii) Cloning of Lower Pathway and Promoter

MVK-PMK-MVD-IDI genes were amplified from pTrcKKDyIkIS with primersMCM104 and MCM105 using Roche Expand Long PCR System according to themanufacturer's instructions. This product was digested with NotI andApaI and cloned into MCM281 which had been digested with NotI and ApaIand gel purified. Primers MCM120 and MCM127 were used to amplify CMRcassette from the GeneBridges FRT-gb2-Cm-FRT template DNA usingStratagene Pfu Ultra II. A PCR program of denaturing at 95° C. for 4:00,5 cycles of 95° C. for 0:20, 55° C. for 0:20, 72° C. for 2:00, 25 cyclesof 95° C. for 0:20, 58° C. for 0:20, 72° C. for 2:00, 72° C. for 10:00,and then cooling to 4° C. was used with four 50 uL PCR reactionscontaining 1 uL ˜10 ng/uL template, 1 uL each primer, 1.25 uL 10 mMdNTPs, 5 uL 10× buffer, 1 uL enzyme, and 39.75 uL ddH20. Reactions werepooled, purified on a Qiagen PCR cleanup column, and used toelectroporate water-washed Pir1 cells containing plasmid MCM296.Electroporation was carried out in 2 mM cuvettes at 2.5V and 200 ohms.Electroporation reactions were recovered in LB for 3 hr at 30° C.Transformant MCM330 was selected on LA with CMPS, Kan50 (FIGS. 107 and108A-108C).

iii) Integration into E. coli Chromosome

Miniprepped DNA (Qiaquick Spin kit) from MCM330 was digested with SnaBIand used to electroporate BL21(DE3) (Novagen) or MG1655 containingGeneBridges plasmid pRedET Carb. Cells were grown at 30° C. to ˜OD1 theninduced with 0.4% L-arabinose at 37° C. for 1.5 hours. These cells werewashed three times in 4° C. ddH2O before electroporation with 2 uL ofDNA. Integrants were selected on L agar with containing chloramphenicol(5 ug/ml) and subsequently confirmed to not grow on L agar+Kanamycin (50ug/ml). BL21 integrant MCM331 and MG1655 integrant MCM333 were frozen.

iv) Construction of pET24D-Kudzu Encoding Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was subcloned into the pET24d vector(Novagen) from the pCR2.1 vector (Invitrogen). In particular, the kudzuisoprene synthase gene was amplified from the pTrcKudzu template DNAusing primers MCM50 5′-GATCATGCAT TCGCCCTTAG GAGGTAAAAA AACATGTGTGCGACCTCTTC TCAATTTACT (SEQ ID NO:99) and MCM53 5′-CGGTCGACGG ATCCCTGCAGTTAGACATAC ATCAGCTG (SEQ ID NO:100). PCR reactions were carried outusing Taq DNA Polymerase (Invitrogen), and the resulting PCR product wascloned into pCR2.1-TOPO TA cloning vector (Invitrogen), and transformedinto E. coli Top10 chemically competent cells (Invitrogen).Transformants were plated on L agar containing carbenicillin (50 μg/ml)and incubated overnight at 37° C. Five ml Luria Broth culturescontaining carbenicillin 50 μg/ml were inoculated with singletransformants and grown overnight at 37° C. Five colonies were screenedfor the correct insert by sequencing of plasmid DNA isolated from 1 mlof liquid culture (Luria Broth) and purified using the QIAprep SpinMini-prep Kit (Qiagen). The resulting plasmid, designated MCM93,contains the kudzu isoprene synthase coding sequence in a pCR2.1backbone.

The kudzu coding sequence was removed by restriction endonucleasedigestion with PciI and BamHI (Roche) and gel purified using theQIAquick Gel Extraction kit (Qiagen). The pET24d vector DNA was digestedwith NcoI and BamHI (Roche), treated with shrimp alkaline phosphatase(Roche), and purified using the QIAprep Spin Mini-prep Kit (Qiagen). Thekudzu isoprene synthase fragment was ligated to the NcoI/BamII1 digestedpET24d using the Rapid DNA Ligation Kit (Roche) at a 5:1 fragment tovector ratio in a total volume of 20 μl. A portion of the ligationmixture (5 μl) was transformed into E. coli Top 10 chemically competentcells and plated on L agar containing kanamycin (50 μg/ml). The correcttransformant was confirmed by sequencing and transformed into chemicallycompetent BL21(λDE3)pLysS cells (Novagen). A single colony was selectedafter overnight growth at 37° C. on L agar containing kanamycin (50μg/ml). A map of the resulting plasmid designated as pET24D-Kudzu isshown in FIG. 109. The sequence of pET24D-Kudzu (SEQ ID NO:101) is shownin FIGS. 110A and 110B. Isoprene synthase polypeptide activity wasconfirmed using a headspace assay.

v) Production Strains

Strains MCM331 and MCM333 were cotransformed with plasmidspCLPtrcupperpathway and either pTrcKudzu or pETKudzu, resulting in thestrains shown in Table 12.

TABLE 12 Production Strains Isoprene Integrated Upper MVA synthaseProduction Background Lower plasmid plasmid Stain BL21(DE3) MCM331pCLPtrcUpper pTrcKudzu MCM343 Pathway BL21(DE3) MCM331 pCLPtrcUpperpET24D- MCM335 Pathway Kudzu MG1655 MCM333 pCLPtrcUpper pTrcKudzu MCM345Pathwayvi) Isoprene Fermentation from E. coli Expressing Genes from theMevalonic Acid Pathway and Grown in Fed-Batch Culture at the 15-L Scale.Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the gi1.2 integrated lower MVA pathway described aboveand the pCL PtrcUpperMVA and pTrcKudzu plasmids. This experiment wascarried out to monitor isoprene formation from glucose at the desiredfermentation pH 7.0 and temperature 30° C. An inoculum of E. coli straintaken from a frozen vial was streaked onto an LB broth agar plate (withantibiotics) and incubated at 37° C. A single colony was inoculated intotryptone-yeast extract medium. After the inoculum grew to OD 1.0,measured at 550 nm, 500 mL was used to inoculate a 15-L bioreactorcontaining an initial working volume of 5 L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time, the glucose feed was decreased tomeet metabolic demands. The total amount of glucose delivered to thebioreactor during the 57 hour fermentation was 3.9 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 100 uMwhen the carbon dioxide evolution rate reached 100 mmol/L/hr. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 111A. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.6 g/L (FIG. 111B). The specificproductivity of isoprene over the course of the fermentation is shown inFIG. 111C and peaked at 1.2 mg/OD/hr. The total amount of isopreneproduced during the 57 hour fermentation was 16.2 g. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 0.9%. The weight percent yield of isoprene from glucose was 0.4%.

Example 18 Construction of the Upper and Lower MVA Pathway forIntegration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B.subtilis under control of the aprE promoter. The upper pathway consistsof two genes; mvaE, which encodes for AACT and HMGR, and mvaS, whichencodes for HMGS. The two genes are fused together with a stop codon inbetween, an RBS site in front of mvaS, and are under the control of theaprE promoter. A terminator is situated after the mvaE gene. Thechloramphenicol resistance marker is cloned after the mvaE gene and theconstruct is integrated at the aprE locus by double cross over usingflanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangsthat will allowed them to be fused together by a PCR reaction. PCRamplifications are carried out using Herculase polymerase according tomanufacturer's instructions.

1. PaprE

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE (SEQ IDNO: 83) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAATemplate: Bacillus subtilis Chromosomal DNA2. mvaE

CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG (SEQ ID NO: 84) CF 07-62(−) Fuse mvaE to mvaS with RBS in between5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTTTCTTAAATC (SEQ ID NO:35)Template: Enterococcus faecalis Chromosomal DNA (from ATCC)3. mvaS

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGGATTGATAAA (SEQ ID NO:36) CF 07-124 (−) Fuse the end of mvaS to the terminator5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGT (SEQ ID NO: 85)Template: Enterococcus faecalis Chromosomal DNA4. B. amyliquefaciens Alkaline Serine Protease Terminator

CF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 136)5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCG CF 07-46 (−) End of B.amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: Bacillus amyliquefaciens Chromosomal DNAPCR Fusion Reactions5. Fuse mvaE to mvaS

CF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon) (SEQ IDNO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTG CF 07-124 (−) Fusethe end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: #2 and 3 from Above6. Fuse mvaE-mvaS to aprE Promoter

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-124 (−) Fuse the end of mvaS to theterminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate #1 and #4 from Above7. Fuse PaprE-mvaE-mvaS to Terminator

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-46 (−) End of B.amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI andligated to pJM102 (Perego, M. 1993. Integrational vectors for geneticmanipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J.A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positivebacteria: biochemistry, physiology, and molecular genetics. AmericanSociety for Microbiology, Washington, D.C.) which is digested withPstI/BamHI. The ligation is transformed into E. coli TOP 10 chemicallycompetent cells and transformants are selected on LA containingcarbenicillin (50 μg/ml). The correct plasmid is identified bysequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51A-51C).Purified plasmid DNA is transformed into Bacillus subtilis aprEnprEPxyl-comK and transformants are selected on L agar containingchloramphenicol (5 μg/ml). A correct colony is selected and is platedsequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml toamplify the number of copies of the cassette containing the upperpathway.

The resulting strain is tested for mevalonic acid production by growingin LB containing 1% glucose and 1%. Cultures are analyzed by GC for theproduction of mevalonic acid.

This strain is used subsequently as a host for the integration of thelower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing Primers:

CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-58 (+) Start of mvaE gene (SEQ IDNO: 38) 5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene (SEQID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGC CF 07-82 (+) Start ofmvaS gene (SEQ ID NO: 40) 5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) Endof mvaS gene (SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGT CF 07-86 (+)Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATC CF 07-87 (+)Sequence in mvaE (SEQ ID NO: 43) 5′-TTGCCAATCATATGATTGAAAATC CF 07-88(+) Sequence in mvaE (SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF07-89 (+) Sequence mvaS (SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at aconcentration of 5 μg/ml. One colony is confirmed to have the correctintegration by sequencing and is plated on LA containing increasingconcentrations of chloramphenicol over several days, to a final level of25 μg/ml. This results in amplification of the cassette containing thegenes of interest. The resulting strain is designated CF 455:pJMupperpathway#1 X Bacillus subtilis aprEnprE Pxyl comK (amplified togrow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idiare combined in a cassette consisting of flanking DNA regions from thenprE region of the B. subtilis chromosome (site of integration), theaprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and29A-29D). This cassette is synthesized by DNA2.0 and is integrated intothe chromosome of B. subtilis containing the upper MVA pathwayintegrated at the aprE locus. The kudzu isoprene synthase gene isexpressed from the replicating plasmid described in Example 13 and istransformed into the strain with both upper and lower pathwaysintegrated.

Example 19 The De-Coupling of Growth and Production of Isoprene in E.coli Expressing Genes from the Mevalonic Acid Pathway and Fermented in aFed-Batch Culture

This example illustrates the de-coupling of cell growth from mevalonicacid and isoprene production.

I. Fermentation Conditions

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH2O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl2.6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in Di H₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume, and filtersterilized with a 0.22 micron filter.

Fermentation was performed with E. coli cells containing thepTrcHis2AUpperPathway (also called pTrcUpperMVA, FIGS. 91 and 92A-92C)(50 μg/ml carbenicillin) or the pCL PtrcUpperMVA (also called pCLPtrcUpperPathway (FIG. 26)) (50 μg/ml spectinomycin) plasmids. Forexperiments in which isoprene was produced, the E. coli cells alsocontained the pTrc KKDyIkIS (50 μg/ml kanamycin) plasmid. Theseexperiments were carried out to monitor mevalonic acid or isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. An inoculum of an E. coli strain taken from a frozenvial was streaked onto an LA broth agar plate (with antibiotics) andincubated at 37° C. A single colony was inoculated into tryptone-yeastextract medium. After the inoculum grew to optical density 1.0 whenmeasured at 550 nm, it was used to inoculate the bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. Induction was achieved by adding IPTG. The mevalonicacid concentration in fermentation broth was determined by applyingperchloric acid (Sigma-Aldrich #244252) treated samples (0.3 M incubatedat 4° C. for 5 minutes) to an organic acids HPLC column (BioRad#125-0140). The concentration was determined by comparing the brothmevalonic acid peak size to a calibration curve generated frommevalonolacetone (Sigma-Aldrich #M4667) treated with perchloric acid toform D,L-mevalonate. The isoprene level in the off gas from thebioreactor was determined as described herein. The isoprene titer isdefined as the amount of isoprene produced per liter of fermentationbroth.

II. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 150-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 19, part I were inoculated into a flask containing 45 mL oftryptone-yeast extract medium and incubated at 30° C. with shaking at170 rpm for 5 hours. This solution was transferred to a 5-L bioreactorof tryptone-yeast extract medium, and the cells were grown at 30° C. and27.5 rpm until the culture reached an OD₅₅₀ of 1.0. The 5 L of inoculumwas seeded into a 150-L bioreactor containing 45-kg of medium. The IPTGconcentration was brought to 1.1 mM when the OD₅₅₀ reached a value of10. The OD₅₅₀ profile within the bioreactor over time is shown in FIG.60A. The mevalonic acid titer increased over the course of thefermentation to a final value of 61.3 g/L (FIG. 60B). The specificproductivity profile throughout the fermentation is shown in FIG. 60Cand a comparison to FIG. 60A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 52.5 hour fermentation was 4.0 kg from 14.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 34.2%.

III. Mevalonic Acid Production from E. coli BL21 (DE3) Cells Expressingthe pTrcUpperMVA Plasmid at a 15-L Scale

BL21 (DE3) cells that were grown on a plate as explained above inExample 19, part I were inoculated into a flask containing 500 mL oftryptone-yeast extract medium and grown at 30° C. at 160 rpm to OD₅₅₀1.0. This material was seeded into a 15-L bioreactor containing 4.5-kgof medium. The IPTG concentration was brought to 1.0 mM when the OD₅₅₀reached a value of 10. The OD₅₅₀ profile within the bioreactor over timeis shown in FIG. 61A. The mevalonic acid titer increased over the courseof the fermentation to a final value of 53.9 g/L (FIG. 61B). Thespecific productivity profile throughout the fermentation is shown inFIG. 61C and a comparison to FIG. 61A illustrates the de-coupling ofgrowth and mevalonic acid production. The total amount of mevalonic acidproduced during the 46.6 hour fermentation was 491 g from 2.1 kg ofutilized glucose. The molar yield of utilized carbon that went intoproducing mevalonic acid during fermentation was 28.8%.

IV. Mevalonic Acid Production from E. coli FM5 Cells Expressing thepTrcUpperMVA Plasmid at a 15-L Scale

FM5 cells that were grown on a plate as explained above in Example 19,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 1.0 mM when the OD₅₅₀ reached avalue of 30. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 62A. The mevalonic acid titer increased over the course of thefermentation to a final value of 23.7 g/L (FIG. 62B). The specificproductivity profile throughout the fermentation is shown in FIG. 62Cand a comparison to FIG. 62A illustrates the de-coupling of growth andmevalonic acid production. The total amount of mevalonic acid producedduring the 51.2 hour fermentation was 140 g from 1.1 kg of utilizedglucose. The molar yield of utilized carbon that went into producingmevalonic acid during fermentation was 15.2%.

V. Isoprene Production from E. coli BL21 (DE3) Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 19,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 25 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 uM when OD₅₅₀reached 190. The IPTG concentration was raised to 100 uM at 38 hours offermentation. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 63A. The isoprene titer increased over the course of thefermentation to a final value of 2.2 g/L broth (FIG. 63B). The specificproductivity profile throughout the fermentation is shown in FIG. 63Cand a comparison to FIG. 63A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the54.4 hour fermentation was 15.9 g from 2.3 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.53%.

VI. Isoprene Production from E. coli BL21 (DE3) Tuner Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

BL21 (DE3) tuner cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 19,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 26 μM when the OD₅₅₀ reached avalue of 10. The IPTG concentration was raised to 50 μM when OD₅₅₀reached 175. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 64A. The isoprene titer increased over the course of thefermentation to a final value of 1.3 g/L broth (FIG. 64B). The specificproductivity profile throughout the fermentation is shown in FIG. 64Cand a comparison to FIG. 64A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the48.6 hour fermentation was 9.9 g from 1.6 kg of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 1.34%.

VII. Isoprene Production from E. coli MG1655 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 19, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 24 μM when the OD₅₅₀ reached a value of 45.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 65A.The isoprene titer increased over the course of the fermentation to afinal value of 393 mg/L broth (FIG. 65B). The specific productivityprofile throughout the fermentation is shown in FIG. 65C and acomparison to FIG. 65A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the67.4 hour fermentation was 2.2 g from 520 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.92%.

VIII. Isoprene Production from E. coli MG1655ack-pta Cells Expressingthe pCL PtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

MG1655ack-pta cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkISplasmids that were grown on a plate as explained above in Example 19,part I were inoculated into a flask containing 500 mL of tryptone-yeastextract medium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. Thismaterial was seeded into a 15-L bioreactor containing 4.5-kg of medium.The IPTG concentration was brought to 30 μM when the OD₅₅₀ reached avalue of 10. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 66A. The isoprene titer increased over the course of thefermentation to a final value of 368 mg/L broth (FIG. 66B). The specificproductivity profile throughout the fermentation is shown in FIG. 66Cand a comparison to FIG. 66A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the56.7 hour fermentation was 1.8 g from 531 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.73%.

IX. Isoprene Production from E. coli FM5 Cells Expressing the pCLPtrcUpperMVA and pTrc KKDyIkIS Plasmids at a 15-L Scale

FM5 cells expressing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmidsthat were grown on a plate as explained above in Example 19, part I wereinoculated into a flask containing 500 mL of tryptone-yeast extractmedium and grown at 30° C. at 160 rpm to OD₅₅₀ 1.0. This material wasseeded into a 15-L bioreactor containing 4.5-kg of medium. The IPTGconcentration was brought to 27 μM when the OD₅₅₀ reached a value of 15.The OD₅₅₀ profile within the bioreactor over time is shown in FIG. 67A.The isoprene titer increased over the course of the fermentation to afinal value of 235 mg/L broth (FIG. 67B). The specific productivityprofile throughout the fermentation is shown in FIG. 67C and acomparison to FIG. 67A illustrates the de-coupling of growth andisoprene production. The total amount of isoprene produced during the52.3 hour fermentation was 1.4 g from 948 g of utilized glucose. Themolar yield of utilized carbon that went into producing isoprene duringfermentation was 0.32%.

Example 20 Production of Isoprene During the Exponential Growth Phase ofE. coli Expressing Genes from the Mevalonic Acid Pathway and Fermentedin a Fed-Batch Culture

This example illustrates the production of isoprene during theexponential growth phase of cells.

Medium Recipe (Per Liter Fermentation Medium):

The medium was generated using the following components per literfermentation medium: K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acidmonohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract 0.5 g, and1000× modified trace metal solution 1 ml. All of the components wereadded together and dissolved in diH₂O. This solution was autoclaved. ThepH was adjusted to 7.0 with ammonium hydroxide (30%) and q.s. to volume.Glucose 10 g, thiamine*HCl 0.1 g, and antibiotics were added aftersterilization and pH adjustment.

1000× Modified Trace Metal Solution:

The 1000× modified trace metal solution was generated using thefollowing components: citric acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g,FeSO₄*7H₂O 1 g, CoCl2*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃100 mg, and NaMoO₄*2H₂O 100 mg. Each component is dissolved one at atime in Di H2O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with ATCC11303 E. colicells containing the pCL PtrcUpperMVA and pTrc KKDyIkIS plasmids. Thisexperiment was carried out to monitor isoprene formation from glucose atthe desired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was streaked onto an LB broth agarplate (with antibiotics) and incubated at 37° C. A single colony wasinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-Lbioreactor containing an initial working volume of 5 L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 50 hour fermentation was 2.0 kg. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 25 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 10. TheIPTG concentration was raised to 50 uM when OD₅₅₀ reached 190. The OD₅₅₀profile within the bioreactor over time is shown in FIG. 99. Theisoprene level in the off gas from the bioreactor was determined asdescribed herein. The isoprene titer increased over the course of thefermentation to a final value of 1.4 g/L (FIG. 100). The total amount ofisoprene produced during the 50 hour fermentation was 10.0 g. Theprofile of the isoprene specific productivity over time within thebioreactor is shown in FIG. 101. The molar yield of utilized carbon thatcontributed to producing isoprene during fermentation was 1.1%. Theweight percent yield of isoprene from glucose was 0.5%.

Example 21 Flammability Modeling and Testing of Isoprene

I. Summary of Flammability Modeling and Testing of Isoprene

Flammability modeling and experiments were performed for varioushydrocarbon/oxygen/nitrogen/water/carbon dioxide mixtures. This modelingand experimental tested was aimed at defining isoprene andoxygen/nitrogen flammability curves under specified steam and carbonmonoxide concentrations at a fixed pressure and temperature. A matrix ofthe model conditions is shown in Table 13, and a matrix of theexperiments performed is shown in Table 14.

TABLE 13 Summary of Modeled Isoprene Flammability Carbon Steam DioxideIsoprene Oxygen Temperature Pressure Concentration ConcentrationConcentration Concentration Series (° C.) (psig) (wt %) (wt. %) (vol. %)(vol. %) A 40 0 0 0 Varying Varying B 40 0 4 0 Varying Varying C 40 0 05 Varying Varying D 40 0 0 10 Varying Varying E 40 0 0 15 VaryingVarying F 40 0 0 20 Varying Varying G 40 0 0 30 Varying Varying

TABLE 14 Summary of Isoprene Flammability Tests Steam Isoprene OxygenTemperature Pressure Concentration Concentration Concentration SeriesNumber (° C.) (psig) (vol. %) (vol. %) (vol. %) 1 40 0 0 Varying Varying2 40 0 4 Varying VaryingII. Description of Calculated Adiabatic Flame Temperature (CAFT) Model

Calculated adiabatic flame temperatures (CAFT) along with a selectedlimit flame temperature for combustion propagation were used todetermine the flammability envelope for isoprene. The computer programused in this study to calculate the flame temperatures is the NASA GlennResearch Center CEA (Chemical Equilibrium with Applications) software.

There are five steps involved in determining the flammability envelopeusing an adiabatic flame temperature model for a homogeneous combustionmechanism (where both the fuel and oxidant are in the gaseous state):selection of the desired reactants, selection of the test condition,selection of the limit flame temperature, modification of the reactants,and construction of a flammability envelope from calculations.

In this first step, selection of desired reactants, a decision must bemade as to the reactant species that will be present in the system andthe quantities of each. In many cases the computer programs used for thecalculations have a list of reactant and product species. If any of thedata for the species to be studied are not found in the program, theymay be obtained from other sources such as the JANAF tables or from theinternet. In this current model data for water, nitrogen, oxygen andcarbon dioxide were present in the program database. The programdatabase did not have isoprene as a species; therefore thermodynamicproperties were incorporated manually.

The next step is to decide whether the initial pressure and temperatureconditions that the combustion process is taking place in. In this modelthe pressure was 1 atmosphere (absolute) and the temperature was 40° C.,the boiling point of isoprene.

The limit flame temperature for combustion can be either selected basedon theoretical principles or determined experimentally. Each method hasits own limitations.

Based on prior studies, the limit flame temperatures of hydrocarbonsfall in the range of 1000 K to 1500 K. For this model, the value of 1500K was selected. This is the temperature at which the reaction of carbonmonoxide to carbon dioxide (a highly exothermic reaction and constitutesa significant proportion of the flame energy) becomes self sustaining.

Once the limit flame temperature has been decided upon, modelcalculations are performed on the given reactant mixture (speciesconcentrations) and the adiabatic flame temperature is determined. Flamepropagation is considered to have occurred only if the temperature isgreater than the limit flame temperature. The reactant mixturecomposition is then modified to create data sets for propagation andnon-propagation mixtures.

This type of model shows good agreement with the experimentallydetermined flammability limits. Regions outside the derived envelope arenonflammable and regions within it are flammable. The shape of theenvelope forms a nose. The nose of the envelope is related to thelimiting oxygen concentration (LOC) for gaseous fuels.

III. Results from Calculated Adiabatic Flame Temperature (CAFT) Model

Plotted in FIGS. 68 through 74 are the CAFT model results for Series Ato G, respectively. The figures plot the calculated adiabatic flametemperature (using the NASA CEA program) as a function of fuelconcentration (by weight) for several oxygen/nitrogen ratios (byweight). The parts of the curve that are above 1500 K, the selectedlimit flame temperature, contain fuel levels sufficient for flamepropagation. The results may be difficult to interpret in the formpresented in FIGS. 68 through 74. Additionally, the current form is notconducive to comparison with experimental data which is generallypresented in terms of volume percent.

Using Series A as an example the data in FIG. 68 can be plotted in theform of a traditional flammability envelope. Using FIG. 68 and readingacross the 1500 K temperature line on the ordinate one can determine thefuel concentration for this limit flame temperature by dropping atangent to the abscissa for each curve (oxygen to nitrogen ratio) thatit intersects. These values can then be tabulated as weight percent offuel for a given weight percent of oxidizer (FIG. 75A). Then knowing thecomposition of the fuel (100 wt. % isoprene) and the composition of theoxidizer (relative content of water, oxygen and nitrogen) molarquantities can be established.

From these molar quantities percentage volume concentrations can becalculated. The concentrations in terms of volume percent can then beplotted to generate a flammability envelope (FIG. 75B). The area boundedby the envelope is the explosible range and the area excluded is thenon-explosible range. The “nose” of the envelope is the limiting oxygenconcentration. FIGS. 76A and 76B contain the calculated volumeconcentrations for the flammability envelope for Series B generated fromdata presented in FIG. 69. A similar approach can be used on datapresented in FIGS. 70-74.

IV. Flammability Testing Experimental Equipment and Procedure

Flammability testing was conducted in a 4 liter high pressure vessel.The vessel was cylindrical in shape with an inner diameter of 6″ and aninternal height of 8.625″. The temperature of the vessel (and the gasesinside) was maintained using external heaters that were controlled by aPID controller. To prevent heat losses, ceramic wool and reflectiveinsulation were wrapped around the pressure vessel. Type K thermocoupleswere used the measure the temperature of the gas space as well as thetemperature of the vessel itself. FIG. 77 illustrates the test vessel.

Before a test was ran, the vessel was evacuated and purged with nitrogento ensure that any gases from previous tests were removed. A vacuum wasthen pulled on the vessel. The pressure after this had been done wastypically around 0.06 bar(a). Due to the nitrogen purging, the gasresponsible for this initial pressure was assumed to be nitrogen. Usingpartial pressures, water, isoprene, nitrogen, and oxygen were then addedin the appropriate amounts to achieve the test conditions in question. Amagnetically driven mixing fan within the vessel ensured mixing of thegaseous contents. The gases were allowed to mix for about 2 minutes withthe fan being turned off approximately 1 minute prior to ignition.

The igniter was comprised of a 1.5 ohm nicrome coil and an AC voltagesource on a timer circuit. Using an oscilloscope, it was determined that34.4 VAC were delivered to the igniter for 3.2 seconds. A maximumcurrent of 3.8 amps occurred approximately halfway into the ignitioncycle. Thus, the maximum power was 131 W and the total energy providedover the ignition cycle was approximately 210 J.

Deflagration data was acquired using a variable reluctance ValidyneDP215 pressure transducer connected to a data acquisition system. A gasmixture was considered to have deflagrated if the pressure rise wasgreater than or equal to 5%.

V. Results of Flammability Testing

The first experimental series (Series 1) was run at 40° C. and 0 psigwith no steam. Running tests at varying concentrations of isoprene andoxygen produced the flammability curve shown in FIG. 78A. The datapoints shown in this curve are only those that border the curve. Adetailed list of all the data points taken for this series is shown inFIGS. 80A and 80B.

FIG. 78B summarizes the explosibility data points shown in FIG. 78A.FIG. 78C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the non-adiabatic natureof the test chamber and limitations of the model. The model looks at aninfinite time horizon for the oxidation reaction and does not take intoconsideration any reaction kinetic limitation.

Additionally, the model is limited by the number of equilibrium chemicalspecies that are in its database and thus may not properly predictpyrolytic species. Also, the flammability envelope developed by themodel uses one value for a limit flame temperature (1500K). The limitflame temperature can be a range of values from 1,000K to 1,500Kdepending on the reacting chemical species. The complex nature ofpyrolytic chemical species formed at fuel concentrations above thestoichiometric fuel/oxidizer level is one reason why the model may notaccurately predict the upper flammable limit for this system.

The second experimental series (Series 2) was run at 40° C. and 0 psigwith a fixed steam concentration of 4%. Running tests at varyingconcentrations of isoprene and oxygen produced the flammability curveshown in FIG. 79A. The data points shown in this curve are only thosethat border the curve. A detailed list of all the data points taken forthis series is shown in FIG. 81. Due to the similarity between the datain Series 1 only the key points of lower flammable limit, limitingoxygen concentration, and upper flammable limits were tested. Theaddition of 4% steam to the test mixture did not significantly changethe key limits of the flammability envelope. It should be noted thathigher concentrations of steam/water and or other inertants mayinfluence the flammability envelope.

FIG. 79B summarizes the explosibility data points shown in FIG. 79A.FIG. 79C is a comparison of the experimental data with the CAFT modelpredicted flammability envelope. The model agrees very well with theexperimental data. Discrepancies may be due to the same factorsdescribed in Series 1

V. Calculation of Flammability Limits of Isoprene in Air at 3Atmospheres of Pressure

The methods described in Example 21, parts I to IV were also used tocalculate the flammability limits of isoprene at an absolute systempressure of 3 atmospheres and 40° C. These results were compared tothose of Example 21, parts I to IV at an absolute system pressure of 1atmosphere and 40° C. This higher pressure was tested because theflammability envelope expands or grows larger as the initial systempressure is increased. The upper flammability limit is affected themost, followed by the limiting oxygen composition. The lowerflammability limit is the least affected (see, for example, “Bulletin627—Flammability Characteristics of Combustible Gases and Vapors”written by Michael G. Zabetakis and published by the former US Bureau ofMines (1965), which is hereby incorporated by reference in its entirety,particular with respect to the calculation of flammability limits).

In FIG. 82, the calculated adiabatic flame temperature is plotted as afunction of isoprene (fuel) concentration, expressed in weight percentof the total fuel/nitrogen/oxygen, where the system pressure wasinitially 3 atmospheres. The calculated flame temperatures are verysimilar to those determined initially in the 1 atmosphere system (FIG.83). As a result, when flammability envelopes are generated using thecalculated adiabatic flammability data, the curves are very similar (seeFIGS. 84 and 85). Therefore, based on these theoretical calculations, asystem pressure increase from 1 atmosphere to 3 atmosphere does notresult in a significant increase/broadening of the flammabilityenvelope. If desired, these model results may be validated usingexperimental testing (such as the experimental testing described hereinat a pressure of 1 atmosphere).VII. Summary of Flammability Studies

A calculated adiabatic temperature model was developed for theflammability envelope of the isoprene/oxygen/nitrogen/water/carbondioxide system at 40° C. and 0 psig. The CAFT model that was developedagreed well with the experimental data generated by the tests conductedin this work. The experimental results from Series 1 and 2 validated themodel results from Series A and B.

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention belongs. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. It is tobe understood that this invention is not limited to the particularmethodology, protocols, and reagents described, as these may vary. Oneof skill in the art will also appreciate that any methods and materialssimilar or equivalent to those described herein can also be used topractice or test the invention.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole.

For use herein, unless clearly indicated otherwise, use of the terms“a”, “an,” and the like refers to one or more.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

APPENDIX 1

Exemplary 1-deoxy-D-xylulose-5-phosphate synthase nucleic acids andpolypeptides

-   ATH: AT3G21500(DXPS1) AT4G15560(CLA1) AT5G11380(DXPS3)-   OSA: 4338768 4340090 4342614-   CME: CMF089C-   PFA: MAL13P1.186-   TAN: TA20470-   TPV: TP01_(—)0516-   ECO: b0420(dxs)-   ECJ: JW0410(dxs)-   ECE: Z0523(dxs)-   ECS: ECs0474-   ECC: c0531(dxs)-   ECI: UTI89_C0443(dxs)-   ECP: ECP_(—)0479-   ECV: APECO1_(—)1590(dxs)-   ECW: EcE24377A_(—)0451(dxs)-   ECX: EcHS_A0491-   STY: STY0461(dxs)-   STT: t2441(dxs)-   SPT: SPA2301(dxs)-   SEC: SC0463(dxs)-   STM: STM0422(dxs)-   YPE: YPO3177(dxs)-   YPK: y1008(dxs)-   YPM: YP_(—)0754(dxs)-   YPA: YPA_(—)2671-   YPN: YPN_(—)0911-   YPP: YPDSF_(—)2812-   YPS: YPTB0939(dxs)-   YPI: YpsIP31758_(—)3112(dxs)-   SFL: SF0357(dxs)-   SFX: S0365(dxs)-   SFV: SFV_(—)0385(dxs)-   SSN: SSON_(—)0397(dxs)-   SBO: SBO_(—)0314(dxs)-   SDY: SDY_(—)0310(dxs)-   ECA: ECA1131(dxs)-   PLU: plu3887(dxs)-   BUC: BU464(dxs)-   BAS: BUsg448(dxs)-   WBR: WGLp144(dxs)-   SGL: SG0656-   KPN: KPN_(—)00372(dxs)-   BFL: Bfl238(dxs)-   BPN: BPEN_(—)244(dxs)-   HIN: HI1439(dxs)-   HIT: NTHI1691(dxs)-   HIP: CGSHiEE_(—)04795-   HIQ: CGSHiGG_(—)01080-   HDU: HD0441(dxs)-   HSO: HS_(—)0905(dxs)-   PMU: PM0532(dxs)-   MSU: MS1059(dxs)-   APL: APL_(—)0207(dxs)-   XFA: XF2249-   XFT: PD1293(dxs)-   XCC: XCC2434(dxs)-   XCB: XC_(—)1678-   XCV: XCV2764(dxs)-   XAC: XAC2565(dxs)-   XOO: XOO2017(dxs)-   XOM: XOO_(—)1900(XOO1900)-   VCH: VC0889-   VVU: VV1_(—)0315-   VVY: VV0868-   VPA: VP0686-   VFI: VF0711-   PPR: PBPRA0805-   PAE: PA4044(dxs)-   PAU: PA14_(—)11550(dxs)-   PAP: PSPA7_(—)1057(dxs)-   PPU: PP_(—)0527(dxs)-   PST: PSPTO_(—)0698(dxs)-   PSB: Psyr_(—)0604-   PSP: PSPPH_(—)0599(dxs)-   PFL: PFL_(—)5510(dxs)-   PFO: Pfl_(—)5007-   PEN: PSEEN0600(dxs)-   PMY: Pmen_(—)3844-   PAR: Psyc_(—)0221(dxs)-   PCR: Pcryo_(—)0245-   ACI: ACIAD3247(dxs)-   SON: SO_(—)1525(dxs)-   SDN: Sden_(—)2571-   SFR: Sfri_(—)2790-   SAZ: Sama_(—)2436-   SBL: Sbal_(—)1357-   SLO: Shew_(—)2771-   SHE: Shewmr4_(—)2731-   SHM: Shewmr7_(—)2804-   SHN: Shewana3_(—)2901-   SHW: Sputw3181_(—)2831-   ILO: IL2138(dxs)-   CPS: CPS_(—)1088(dxs)-   PHA: PSHAa2366(dxs)-   PAT: Patl_(—)1319-   SDE: Sde_(—)3381-   PIN: Ping_(—)2240-   MAQ: Maqu_(—)2438-   MCA: MCA0817(dxs)-   FTU: FTT1018c(dxs)-   FTF: FTF1018c(dxs)-   FTW: FTW_(—)0925(dxs)-   FTL: FTL_(—)1072-   FTH: FTH_(—)1047(dxs)-   FTA: FTA_(—)1131(dxs)-   FTN: FTN_(—)0896(dxs)-   NOC: Noc_(—)1743-   AEH: Mlg_(—)1381-   HCH: HCH_(—)05866(dxs)-   CSA: Csal_(—)0099-   ABO: ABO_(—)2166(dxs)-   AHA: AHA_(—)3321(dxs)-   BCI: BCI_(—)0275(dxs)-   RMA: Rmag_(—)0386-   VOK: COSY_(—)0360(dxs)-   NME: NMB1867-   NMA: NMA0589(dxs)-   NMC: NMCO352(dxs)-   NGO: NG00036-   CVI: CV_(—)2692(dxs)-   RSO: RSc2221(dxs)-   REU: Reut_A0882-   REH: H16_A2732(dxs)-   RME: Rmet_(—)2615-   BMA: BMAA0330(dxs)-   BMV: BMASAVP1_(—)1512(dxs)-   BML: BMA10299_(—)1706(dxs)-   BMN: BMA10247_A0364(dxs)-   BXE: Bxe_B2827-   BUR: Bcep18194_B2211-   BCN: Bcen_(—)4486-   BCH: Bcen2424_(—)3879-   BAM: Bamb_(—)3250-   BPS: BPSS1762(dxs)-   BPM: BURPS1710b_A0842(dxs)-   BPL: BURPS1106A_A2392(dxs)-   BPD: BURPS668_A2534(dxs)-   BTE: BTH_II0614(dxs)-   BPE: BP2798(dxs)-   BPA: BPP2464(dxs)-   BBR: BB1912(dxs)-   RFR: Rfer_(—)2875-   POL: Bpro_(—)1747-   PNA: Pnap_(—)1501-   AJS: Ajs_(—)1038-   MPT: Mpe_A2631-   HAR: HEAR0279(dxs)-   MMS: mma_(—)0331-   NEU: NE1161(dxs)-   NET: Neut_(—)1501-   NMU: Nmul_A0236-   EBA: ebA4439(dxs)-   AZO: azo1198(dxs)-   DAR: Daro_(—)3061-   TBD: Tbd_(—)0879-   MFA: Mfla_(—)2133-   HPY: HP0354(dxs)-   HPJ: jhp0328(dxs)-   HPA: HPAG1_(—)0349-   HHE: HH0608(dxs)-   HAC: Hac_(—)0968(dxs)-   WSU: WS1996-   TDN: Tmden_(—)0475-   CJE: Cj0321(dxs)-   CJR: CJE0366(dxs)-   CJJ: CJJ81176_(—)0343(dxs)-   CJU: C8J_(—)0298(dxs)-   CJD: JJD26997_(—)1642(dxs)-   CFF: CFF8240_(—)0264(dxs)-   CCV: CCV52592_(—)1671(dxs) CCV52592_(—)1722-   CHA: CHAB381_(—)1297(dxs)-   CCO: CCC13826_(—)1594(dxs)-   ABU: Abu_(—)2139(dxs)-   NIS: NIS_(—)0391(dxs)-   SUN: SUN_(—)2055(dxs)-   GSU: GSU0686(dxs-1) GSU1764(dxs-2)-   GME: Gmet_(—)1934 Gmet_(—)2822-   PCA: Pear_(—)1667-   PPD: Ppro_(—)1191 Ppro_(—)2403-   DVU: DVU1350(dxs)-   DVL: Dvul_(—)1718-   DDE: Dde_(—)2200-   LIP: LI0408(dsx)-   DPS: DP2700-   ADE: Adeh_(—)1097-   MXA: MXAN_(—)4643(dxs)-   SAT: SYN_(—)02456-   SFU: Sfum_(—)1418-   PUB: SAR11_(—)0611(dxs)-   MLO: mlr7474-   MES: Meso_(—)0735-   SME: SMc00972(dxs)-   ATU: Atu0745(dxs)-   ATC: AGR_C_(—)1351-   RET: RHE_CH00913(dxs)-   RLE: RL0973(dxs)-   BME: BMEI1498-   BMF: BAB1_(—)0462(dxs)-   BMS: BR0436(dxs)-   BMB: BruAb1_(—)0458(dxs)-   BOV: BOV_(—)0443(dxs)-   BJA: b112651(dxs)-   BRA: BRADO2161(dxs)-   BBT: BBta_(—)2479(dxs)-   RPA: RPA0952(dxs)-   RPB: RPB_(—)4460-   RPC: RPC_(—)1149-   RPD: RPD_(—)4305-   RPE: RPE_(—)1067-   NWI: Nwi_(—)0633-   NHA: Nham_(—)0778-   BHE: BH04350(dxs)-   BQU: BQ03540(dxs)-   BBK: BARBAKC583_(—)0400(dxs)-   CCR: CC_(—)2068-   SIL: SPO0247(dxs)-   SIT: TM1040_(—)2920-   RSP: RSP_(—)0254(dxsA) RSP_(—)1134(dxs)-   JAN: Jann_(—)0088 Jann_(—)0170-   RDE: RD1_(—)0101(dxs) RD1_(—)0548(dxs)-   MMR: Mmar10_(—)0849-   HNE: HNE_(—)1838(dxs)-   ZMO: ZMO1234(dxs) ZMO1598(dxs)-   NAR: Saro_(—)0161-   SAL: Sala_(—)2354-   ELI: ELI_(—)12520-   GOX: GOX0252-   GBE: GbCGDNIH1_(—)0221 GbCGDNIH1_(—)2404-   RRU: Rru_A0054 Rru_A2619-   MAG: amb2904-   MGM: Mmc1_(—)1048-   SUS: Acid_(—)1783-   BSU: BG11715(dxs)-   BHA: BH2779-   BAN: BA4400(dxs)-   BAR: GBAA4400(dxs)-   BAA: BA_(—)4853-   BAT: BAS4081-   BCE: BC4176(dxs)-   BCA: BCE_(—)4249(dxs)-   BCZ: BCZK3930(dxs)-   BTK: BT9727_(—)3919(dxs)-   BTL: BALH_(—)3785(dxs)-   BLI: BL01523(dxs)-   BLD: BLi02598(dxs)-   BCL: ABC2462(dxs)-   BAY: RBAM_(—)022600-   BPU: BPUM_(—)2159-   GKA: GK2392-   GTN: GTNG_(—)2322-   LMO: lmo1365(tktB)-   LMF: LMOf2365_(—)1382(dxs)-   LIN: lin1402(tktB)-   LWE: lwe1380(tktB)-   LLA: L108911(dxsA) L123365(dxsB)-   LLC: LACR_(—)1572 LACR_(—)1843-   LLM: llmg_(—)0749(dxsB)-   SAK: SAK_(—)0263-   LPL: lp_(—)2610(dxs)-   LJO: LJ0406-   LAC: LBA0356-   LSL: LSL_(—)0209(dxs)-   LGA: LGAS_(—)0350-   STH: STH1842-   CAC: CAC2077 CA_P0106(dxs)-   CPE: CPE1819-   CPF: CPF_(—)2073(dxs)-   CPR: CPR_(—)1787(dxs)-   CTC: CTC01575-   CNO: NT01CX_(—)1983-   CTH: Cthe_(—)0828-   CDF: CD1207(dxs)-   CBO: CBO1881(dxs)-   CBA: CLB_(—)1818(dxs)-   CBH: CLC_(—)1825(dxs)-   CBF: CLI_(—)1945(dxs)-   CKL: CKL_(—)1231(dxs)-   CHY: CHY_(—)1985(dxs)-   DSY: DSY2348-   DRM: Dred_(—)1078-   PTH: PTH_(—)1196(dxs)-   SWO: Swol_(—)0582-   CSC: Csac_(—)1853-   TTE: TTE1298(dxs)-   MTA: Moth_(—)1511-   MPE: MYPE730-   MGA: MGA_(—)1268(dxs)-   MTU: Rv2682c(dxs1) Rv3379c(dxs2)-   MTC: MT2756(dxs)-   MBO: Mb2701c(dxs1) Mb3413c(dxs2)-   MLE: ML1038(dxs)-   MPA: MAP2803c(dxs)-   MAV: MAV_(—)3577(dxs)-   MSM: MSMEG_(—)2776(dxs)-   MMC: Mmcs_(—)2208-   CGL: NCg11827(cg11902)-   CGB: cg2083(dxs)-   CEF: CE1796-   CDI: DIP1397(dxs)-   CJK: jk1078(dxs)-   NFA: nfa37410(dxs)-   RHA: RHA1_ro06843-   SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17)-   SMA: SAV1646(dxs1) SAV2244(dxs2)-   TWH: TWT484-   TWS: TW280(Dxs)-   LXX: Lxx10450(dxs)-   CMI: CMM_(—)1660(dxsA)-   AAU: AAur_(—)1790(dxs)-   PAC: PPA1062-   TFU: Tfu_(—)1917-   FRA: Francci3_(—)1326-   FAL: FRAAL2088(dxs)-   ACE: Acel_(—)1393-   SEN: SACE_(—)1815(dxs) SACE_(—)4351-   BLO: BL1132(dxs)-   BAD: BAD_(—)0513(dxs)-   FNU: FN1208 FN1464-   RBA: RB2143(dxs)-   CTR: CT331(dxs)-   CTA: CTA_(—)0359(dxs)-   CMU: TC0608-   CPN: CPn1060(tktB_(—)2)-   CPA: CP0790-   CPJ: CPj1060(tktB_(—)2)-   CPT: CpB1102-   CCA: CCA00304(dxs)-   CAB: CAB301(dxs)-   CFE: CF0699(dxs)-   PCU: pc0619(dxs)-   TPA: TP0824-   TDE: TDE1910(dxs)-   LIL: LA3285(dxs)-   LIC: LIC10863(dxs)-   LBJ: LBJ_(—)0917(dxs)-   LBL: LBL_(—)0932(dxs)-   SYN: sll1945(dxs)-   SYW: SYNW1292(Dxs)-   SYC: syc1087_c(dxs)-   SYF: Synpcc7942_(—)0430-   SYD: Syncc9605_(—)1430-   SYE: Syncc9902_(—)1069-   SYG: sync_(—)1410(dxs)-   SYR: SynRCC307_(—)1390(dxs)-   SYX: SynWH7803_(—)1223(dxs)-   CYA: CYA_(—)1701(dxs)-   CYB: CYB_(—)1983(dxs)-   TEL: tll0623-   GVI: gll0194-   ANA: alr0599-   AVA: Ava_(—)4532-   PMA: Pro0928(dxs)-   PMM: PMM0907(Dxs)-   PMT: PMT0685(dxs)-   PMN: PMN2A_(—)0300-   PMI: PMT9312_(—)0893-   PMB: A9601_(—)09541(dxs)-   PMC: P9515_(—)09901(dxs)-   PMF: P9303_(—)15371(dxs)-   PMG: P9301_(—)09521(dxs)-   PMH: P9215_(—)09851-   PMJ: P9211_(—)08521-   PME: NATL1_(—)09721(dxs)-   TER: Tery_(—)3042-   BTH: BT_(—)1403 BT_(—)4099-   BFR: BF0873 BF4306-   BFS: BF0796(dxs) BF4114-   PGI: PG2217(dxs)-   CHU: CHU_(—)3643(dxs)-   GFO: GFO_(—)3470(dxs)-   FPS: FP0279(dxs)-   CTE: CT0337(dxs)-   CPH: Cpha266_(—)0671-   PVI: Cvib_(—)0498-   PLT: Plut_(—)0450-   DET: DET0745(dxs)-   DEH: cbdb_A720(dxs)-   DRA: DR_(—)1475-   DGE: Dgeo_(—)0994-   TTH: TTC1614-   TTJ: TTHA0006-   AAE: aq_(—)881-   TMA: TM1770-   PMO: Pmob_(—)1001    Exemplary Acetyl-CoA-Acetyltransferase Nucleic Acids and    Polypeptides-   HSA: 38(ACAT1) 39(ACAT2)-   PTR: 451528(ACAT1)-   MCC: 707653(ACAT1) 708750(ACAT2)-   MMU: 110446(Acat1) 110460(Acat2)-   RNO: 25014(Acat1)-   CFA: 484063(ACAT2) 489421(ACAT1)-   GGA: 418968(ACAT1) 421587(RCJMB04_(—)34i5)-   XLA: 379569(MGC69098) 414622(MGC81403) 414639(MGC81256)    444457(MGC83664)-   XTR: 394562(acat2)-   DRE: 30643(acat2)-   SPU: 759502(LOC759502)-   DME: Dmel_CG10932 Dmel_CG9149-   CEL: T02G5.4 T02G5.7 T02G5.8(kat-1)-   ATH: AT5G48230(ACAT2/EMB1276)-   OSA: 4326136 4346520-   CME: CMA042C CME087C-   SCE: YPL028W(ERG10)-   AGO: AGOS_ADR165C-   PIC: PICST_(—)31707(ERG10)-   CAL: Ca019.1591(erg10)-   CGR: CAGL0L12364g-   SPO: SPBC215.09c-   MGR: MGG_(—)01755 MGG_(—)13499-   ANI: AN1409.2-   AFM: AFUA_(—)6G14200 AFUA_(—)8G04000-   AOR: AO090103000012 AO090103000406-   CNE: CNCO5280-   UMA: UM03571.1-   DDI: DDB_(—)0231621-   PFA: PF14_(—)0484-   TET: TTHERM_(—)00091590 TTHERM_(—)00277470 TTHERM_(—)00926980-   TCR: 511003.60-   ECO: b2224(atoB)-   ECJ: JW2218(atoB) JW5453(yqeF)-   ECE: Z4164(yqeF)-   ECS: ECs3701-   ECC: c2767(atoB) c3441(yqeF)-   ECI: UTI89_C2506(atoB) UTI89_C3247(yqeF)-   ECP: ECP_(—)2268 ECP_(—)2857-   ECV: APECO1_(—)3662(yqeF) APECO1_(—)4335(atoB) APECO1_(—)43352(atoB)-   ECX: EcHS_A2365-   STY: STY3164(yqeF)-   STT: t2929(yqeF)-   SPT: SPA2886(yqeF)-   SEC: SC2958(yqeF)-   STM: STM3019(yqeF)-   SFL: SF2854(yqeF)-   SFX: 53052(yqeF)-   SFV: SFV_(—)2922(yqeF)-   SSN: SSON_(—)2283(atoB) SSON_(—)3004(yqeF)-   SBO: SBO_(—)2736(yqeF)-   ECA: ECA1282(atoB)-   ENT: Ent638_(—)3299-   SPE: Spro_(—)0592-   HIT: NTHI0932(atoB)-   XCC: XCC1297(atoB)-   XCB: XC_(—)2943-   XCV: XCV1401(th1A)-   XAC: XAC1348(atoB)-   XOO: XOO1881(atoB)-   XOM: XOO_(—)1778(XOO1778)-   VCH: VCA0690-   VCO: VCO395_(—)0630-   VVU: VV2_(—)0494 VV2_(—)0741-   VVY: VVA1043 VVA1210-   VPA: VPA0620 VPA1123 VPA1204-   PPR: PBPRB1112 PBPRB1840-   PAE: PA2001(atoB) PA2553 PA3454 PA3589 PA3925-   PAU: PA14_(—)38630(atoB)-   PPU: PP_(—)2051(atoB) PP_(—)2215(fadAx) PP_(—)3754 PP_(—)4636-   PPF: Pput_(—)2009 Pput_(—)2403 Pput_(—)3523 Pput_(—)4498-   PST: PSPTO_(—)0957(phbA-1) PSPTO_(—)3164(phbA-2)-   PSB: Psyr_(—)0824 Psyr_(—)3031-   PSP: PSPPH_(—)0850(phbA1) PSPPH_(—)2209(phbA2)-   PFL: PFL_(—)1478(atoB-2) PFL_(—)2321 PFL_(—)3066 PFL_(—)4330(atoB-2)    PFL_(—)5283-   PFO: Pfl_(—)1269 Pfl_(—)1739 Pfl_(—)2074 Pfl_(—)2868-   PEN: PSEEN3197 PSEEN3547(fadAx) PSEEN4635(phbA)-   PMY: Pmen_(—)1138 Pmen_(—)2036 Pmen_(—)3597 Pmen_(—)3662    Pmen_(—)3820-   PAR: Psyc_(—)0252 Psyc_(—)1169-   PCR: Pcryo_(—)0278 Pcryo_(—)1236 Pcryo_(—)1260-   PRW: PsycPRwf_(—)2011-   ACI: ACIAD0694 ACIAD1612 ACIAD2516(atoB)-   SON: SO_(—)1677(atoB)-   SDN: Sden_(—)1943-   SFR: Sfri_(—)1338 Sfri_(—)2063-   SAZ: Sama_(—)1375-   SBL: Sbal_(—)1495-   SBM: Shew185_(—)1489-   SBN: Sba1195_(—)1525-   SLO: Shew_(—)1667 Shew_(—)2858-   SPC: Sputcn32_(—)1397-   SSE: Ssed_(—)1473 Ssed_(—)3533-   SPL: Spea_(—)2783-   SHE: Shewmr4_(—)2597-   SHM: Shewmr7_(—)2664-   SHN: Shewana3_(—)2771-   SHW: Sputw3181_(—)2704-   ILO: IL0872-   CPS: CPS_(—)1605 CPS_(—)2626-   PHA: PSHAa0908 PSHAa1454(atoB) PSHAa1586(atoB)-   PAT: Patl_(—)2923-   SDE: Sde_(—)3149-   PIN: Ping_(—)0659 Ping_(—)2401-   MAQ: Maqu_(—)2117 Maqu_(—)2489 Maqu_(—)2696 Maqu_(—)3162-   CBU: CBU_(—)0974-   LPN: lpg1825(atoB)-   LPF: lpl1789-   LPP: lpp1788-   NOC: Noc_(—)1891-   AEH: Mlg_(—)0688 Mlg_(—)2706-   HHA: Hhal_(—)1685-   HCH: HCH_(—)05299-   CSA: Csal_(—)0301 Csal_(—)3068-   ABO: ABO_(—)0648(fadAx)-   MMW: Mmwyll_(—)0073 Mmwyll_(—)3021 Mmwyll_(—)3053 Mmwyll_(—)3097    Mmwyll_(—)4182-   AHA: AHA_(—)2143(atoB)-   CVI: CV_(—)2088(atoB) CV_(—)2790(phaA)-   RSO: RSc0276(atoB) RSc1632(phbA) RSc1637(bktB) RSc1761(RS02948)-   REU: Reut_A0138 Reut_A1348 Reut_A1353 Reut_B4561 Reut_B4738

Reut_B5587 Reut_C5943 Reut_C6062

-   REH: H16_A0170 H16_A0867 H16_A0868 H16_A0872 H16_A1297

H16_A1438(phaA) H16_A1445(bktB) H16_A1528 H16_A1713 H16_A1720

H16_A1887 H16_A2148 H16_B0380 H16_B0381 H16_B0406 H16_B0662

H16_B0668 H16_B0759 H16_B1369 H16_B1771

-   RME: Rmet_(—)0106 Rmet_(—)1357 Rmet_(—)1362 Rmet_(—)5156-   BMA: BMA1316 BMA1321(phbA) BMA1436-   BMV: BMASAVP1_A1805(bktB) BMASAVP1_A1810(phbA)-   BML: BMA10299_A0086(phbA) BMA10299_A0091-   BMN: BMA10247_(—)1076(bktB) BMA10247_(—)1081(phbA)-   BXE: Bxe_A2273 Bxe_A2335 Bxe_A2342 Bxe_A4255 Bxe_B0377 Bxe_B0739

Bxe_C0332 Bxe_C0574 Bxe_C0915

-   BVI: Bcep1808_(—)0519 Bcep1808_(—)1717 Bcep1808_(—)2877    Bcep1808_(—)3594

Bcep1808_(—)4015 Bcep1808_(—)5507 Bcep1808_(—)5644

-   BUR: Bcep18194_A3629 Bcep18194_A5080 Bcep18194_A5091

Bcep18194_A6102 Bcep18194_B0263 Bcep18194_B1439

Bcep18194_C6652 Bcep18194_C6802 Bcep18194_C6874

Bcep18194_C7118 Bcep18194_C7151 Bcep18194_C7332

-   BCN: Bcen_(—)1553 Bcen_(—)1599 Bcen_(—)2158 Bcen_(—)2563    Bcen_(—)2998 Bcen_(—)6289-   BCH: Bcen2424_(—)0542 Bcen2424_(—)1790 Bcen2424_(—)2772    Bcen2424_(—)5368

Bcen2424_(—)6232 Bcen2424_(—)6276

-   BAM: Bamb_(—)0447 Bamb_(—)1728 Bamb_(—)2824 Bamb_(—)4717    Bamb_(—)5771 Bamb_(—)5969-   BPS: BPSL1426 BPSL1535(phbA) BPSL1540-   BPM: BURPS1710b_(—)2325(bktB) BURPS1710b_(—)2330(phbA)

BURPS1710b_(—)2453(atoB-2)

-   BPL: BURPS1106A_(—)2197(bktB) BURPS1106A_(—)2202(phbA)-   BPD: BURPS668_(—)2160(bktB) BURPS668_(—)2165(phbA)-   BTE: BTH_I2144 BTH_I2256 BTH_I2261-   PNU: Pnuc₁₃ 0927-   BPE: BP0447 BP0668 BP2059-   BPA: BPP0608 BPP1744 BPP3805 BPP4216 BPP4361-   BBR: BB0614 BB3364 BB4250 BB4804 BB4947-   RFR: Rfer_(—)0272 Rfer_(—)1000 Rfer_(—)1871 Rfer_(—)2273    Rfer_(—)2561 Rfer_(—)2594

Rfer_(—)3839

-   POL: Bpro_(—)1577 Bpro_(—)2140 Bpro_(—)3113 Bpro_(—)4187-   PNA: Pnap_(—)0060 Pnap_(—)0458 Pnap_(—)0867 Pnap_(—)1159    Pnap_(—)2136 Pnap_(—)2804-   AAV: Aave_(—)0031 Aave_(—)2478 Aave_(—)3944 Aave_(—)4368-   AJS: Ajs_(—)0014 Ajs_(—)0124 Ajs_(—)1931 Ajs_(—)2073 Ajs_(—)2317    Ajs_(—)3548

Ajs_(—)3738 Ajs_(—)3776

-   VEI: Veis_(—)1331 Veis_(—)3818 Veis_(—)4193-   DAC: Daci_(—)0025 Daci_(—)0192 Daci_(—)3601 Daci_(—)5988-   MPT: Mpe_A1536 Mpe_A1776 Mpe_A1869 Mpe_A3367-   HAR: HEAR0577(phbA)-   MMS: mma_(—)0555-   NEU: NE2262(bktB)-   NET: Neut_(—)0610-   EBA: ebA5202 p2A409(tioL)-   AZO: azo0464(fadA1) azo0469(fadA2) azo2172(th1A)-   DAR: Daro_(—)0098 Daro_(—)3022-   HPA: HPAG1_(—)0675-   HAC: Hac_(—)0958(atoB)-   GME: Gmet_(—)1719 Gmet_(—)2074 Gmet_(—)2213 Gmet_(—)2268    Gmet_(—)3302-   GUR: Gura_(—)3043-   BBA: Bd0404(atoB) Bd2095-   DOL: Dole_(—)0671 Dole_(—)1778 Dole_(—)2160 Dole_(—)2187-   ADE: Adeh_(—)0062 Adeh_(—)2365-   AFW: Anae109_(—)0064 Anae109_(—)1504-   MXA: MXAN_(—)3791-   SAT: SYN_(—)02642-   SFU: Sfum_(—)2280 Sfum_(—)3582-   RPR: RP737-   RCO: RC1134 RC1135-   RFE: RF_(—)0163(paaJ)-   RBE: RBE_(—)0139(paaJ)-   RAK: A1C_(—)05820-   RBO: A1I_(—)07215-   RCM: A1E_(—)04760-   PUB: SAR11_(—)0428(th1A)-   MLO: mlr3847-   MES: Meso_(—)3374-   PLA: Play_(—)1573 Play_(—)2783-   SME: SMa1450 SMc03879(phbA)-   SMD: Smed_(—)0499 Smed_(—)3117 Smed_(—)5094 Smed_(—)5096-   ATU: Atu2769(atoB) Atu3475-   ATC: AGR_C_(—)5022(phbA) AGR_L_(—)2713-   RET: RHE_CH04018(phbAch) RHE_PC00068(ypc00040) RHE_PF00014(phbAf)-   RLE: RL4621(phaA) pRL100301 pRL120369-   BME: BMEI0274 BMEII0817-   BMF: BAB1_(—)1783(phbA-1) BAB2_(—)0790(phbA-2)-   BMS: BR1772(phbA-1) BRA0448(phbA-2)-   BMB: BruAb1_(—)1756(phbA-1) BruAb2_(—)0774(phbA-2)-   BOV: BOV_(—)1707(phbA-1)-   OAN: Oant_(—)1130 Oant_(—)3107 Oant_(—)3718 Oant_(—)4020-   BJA: bll0226(atoB) bll3949 bll7400 bll7819 blr3724(phbA)-   BRA: BRADO0562(phbA) BRADO0983(pimB) BRADO3110 BRADO3134(atoB)-   BBT: BBta_(—)3558 BBta_(—)3575(atoB) BBta_(—)5147(pimB)    BBta_(—)7072(pimB)

BBta_(—)7614(phbA)

-   RPA: RPA0513(pcaF) RPA0531 RPA3715(pimB)-   RPB: RPB_(—)0509 RPB_(—)0525 RPB_(—)1748-   RPC: RPC_(—)0504 RPC_(—)0636 RPC_(—)0641 RPC_(—)0832 RPC_(—)1050    RPC_(—)2005

RPC_(—)2194 RPC_(—)2228

-   RPD: RPD_(—)0306 RPD_(—)0320 RPD_(—)3105 RPD_(—)3306-   RPE: RPE_(—)0168 RPE_(—)0248 RPE_(—)3827-   NWI: Nwi_(—)3060-   XAU: Xaut_(—)3108 Xaut_(—)4665-   CCR: CC_(—)0510 CC_(—)0894 CC_(—)3462-   SIL: SPO0142(bktB) SPO0326(phbA) SPO0773 SPO3408-   SIT: TM1040_(—)0067 TM1040_(—)2790 TM1040_(—)3026 TM1040_(—)3735-   RSP: RSP_(—)0745 RSP_(—)1354 RSP_(—)3184-   RSH: Rsph17029_(—)0022 Rsph17029_(—)2401 Rsph17029_(—)3179    Rsph17029_(—)3921-   RSQ: Rsph17025_(—)0012 Rsph17025_(—)2466 Rsph17025_(—)2833-   JAN: Jann_(—)0262 Jann_(—)0493 Jann_(—)4050-   RDE: RD1_(—)0025 RD1_(—)0201(bktB) RD1_(—)3394(phbA)-   PDE: Pden_(—)2026 Pden_(—)2663 Pden_(—)2870 Pden_(—)2907    Pden_(—)4811 Pden_(—)5022-   DSH: Dshi_(—)0074 Dshi_(—)3066 Dshi_(—)3331-   MMR: Mmar10_(—)0697-   HNE: HNE_(—)2706 HNE_(—)3065 HNE_(—)3133-   NAR: Saro_(—)0809 Saro_(—)1069 Saro_(—)1222 Saro_(—)2306    Saro_(—)2349-   SAL: Sala_(—)0781 Sala_(—)1244 Sala_(—)2896 Sala_(—)3158-   SWI: Swit_(—)0632 Swit_(—)0752 Swit_(—)2893 Swit_(—)3602    Swit_(—)4887 Swit_(—)5019-   Swit_(—)5309-   ELI: ELI_(—)01475 ELI_(—)06705 ELI_(—)12035-   GBE: GbCGDNIH1_(—)0447-   ACR: Acry_(—)1847 Acry_(—)2256-   RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946 Rru_A3387-   MAG: amb0842-   MGM: Mmc1_(—)1165-   ABA: Acid345_(—)3239-   BSU: BG11319(mmgA) BG13063(yhfS)-   BHA: BH1997 BH2029 BH3801(mmgA)-   BAN: BA3687 BA4240 BA5589-   BAR: GBAA3687 GBAA4240 GBAA5589-   BAA: BA_(—)0445 BA_(—)4172 BA_(—)4700-   BAT: BAS3418 BAS3932 BAS5193-   BCE: BC3627 BC4023 BC5344-   BCA: BCE_(—)3646 BCE_(—)4076 BCE_(—)5475-   BCZ: BCZK3329(mmgA) BCZK3780(th1) BCZK5044(atoB)-   BCY: Bcer98_(—)2722 Bcer98_(—)3865-   BTK: BT9727_(—)3379(mmgA) BT9727_(—)3765(th1) BT9727_(—)5028(atoB)-   BTL: BALH_(—)3262(mmgA) BALH_(—)3642(fadA) BALH_(—)4843(atoB)-   BLI: BL03925(mmgA)-   BLD: BLi03968(mmgA)-   BCL: ABC0345 ABC2989 ABC3617 ABC3891(mmgA)-   BAY: RBAM_(—)022450-   BPU: BPUM_(—)2374(yhfS) BPUM_(—)2941 BPUM_(—)3373-   OIII: OB0676 OB0689 OB2632 OB3013-   GKA: GK1658 GK3397-   SAU: SA0342 SA0534(vraB)-   SAV: SAV0354 SAV0576(vraB)-   SAM: MW0330 MW0531(vraB)-   SAR: SAR0351(th1) SAR0581-   SAS: SAS0330 SAS0534-   SAC: SACOL0426 SACOL0622(atoB)-   SAB: SAB0304(th1) SAB0526-   SAA: SAUSA300_(—)0355 SAUSA300_(—)0560(vraB)-   SAO: SAOUHSC_(—)00336 SAOUHSC_(—)00558-   SAT: SaurJH9_(—)0402-   SAH: SaurJH1_(—)0412-   SEP: SE0346 SE2384-   SER: SERP0032 SERP0220-   SHA: SH0510(mvaC) SH2417-   SSP: SSP0325 SSP2145-   LMO: lmo1414-   LMF: LMOf2365_(—)1433-   LIN: lin1453-   LWE: lwe1431-   LLA: L11745(thiL) L25946(fadA)-   LLC: LACK_(—)1665 LACK_(—)1956-   LLM: llmg_(—)0930(thiL)-   SPY: SPy_(—)0140 SPy_(—)1637(atoB)-   SPZ: M5005_Spy_(—)0119 M5005_Spy_(—)0432 M5005_Spy_(—)1344(atoB)-   SPM: spyM18_(—)0136 spyM18_(—)1645(atoB)-   SPG: SpyM3_(—)0108 SpyM3_(—)1378(atoB)-   SPS: SPs0110 SPs0484-   SPH: MGAS10270_Spy0121 MGAS10270_Spy0433 MGAS10270_Spy1461(atoB)-   SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 MGAS10750_Spy1453(atoB)-   SPJ: MGAS2096_Spy0123 MGAS2096_Spy0451 MGAS2096_Spy1365(atoB)-   SPK: MGAS9429_Spy0121 MGAS9429_Spy0431 MGAS9429_Spy1339(atoB)-   SPF: SpyM50447(atoB2)-   SPA: M6_Spy0166 M6_Spy0466 M6_Spy1390-   SPB: M28_Spy0117 M28_Spy0420 M28_Spy1385(atoB)-   SAK: SAK_(—)0568-   LJO: LJ1609-   LAC: LBA0626(thiL)-   LSA: LSA1486-   LDB: Ldb0879-   LBU: LBUL_(—)0804-   LBR: LVIS_(—)2218-   LCA: LSEI_(—)1787-   LGA: LGAS_(—)1374-   LRE: Lreu_(—)0052-   EFA: EF1364-   OOE: OEOE_(—)0529-   STH: STH2913 STH725 STH804-   CAC: CAC2873 CA_P0078(thiL)-   CPE: CPE2195(atoB)-   CPF: CPF_(—)2460-   CPR: CPR_(—)2170-   CTC: CTC00312-   CNO: NT01CX_(—)0538 NT01CX_(—)0603-   CDF: CD1059(th1A1) CD2676(th1A2)-   CBO: CBO3200(th1)-   CBE: Cbei_(—)0411 Cbei_(—)3630-   CKL: CKL_(—)3696(th1A1) CKL_(—)3697(th1A2) CKL_(—)3698(th1A3)-   AMT: Amet_(—)4630-   AOE: Clos_(—)0084 Clos_(—)0258-   CHY: CHY_(—)1288 CHY_(—)1355(atoB) CHY_(—)1604 CHY_(—)1738-   DSY: DSY0632 DSY0639 DSY1567 DSY1710 DSY2402 DSY3302-   DRM: Dred_(—)0400 Dred_(—)1491 Dred_(—)1784 Dred_(—)1892-   SWO: Swol_(—)0308 Swol_(—)0675 Swol_(—)0789 Swol_(—)1486    Swol_(—)1934 Swol_(—)2051-   TTE: TTE0549(paaJ)-   MTA: Moth_(—)1260-   MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5)-   MTC: MT1365(phbA)-   MBO: Mb1167 Mb1358(fadA4) Mb3576(fadA5) Mb3586c(fadA6)-   MBB: BCG_(—)1197 BCG_(—)1385(fadA4) BCG_(—)3610(fadA5)    BCG_(—)3620c(fadA6)-   MLE: ML1158(fadA4)-   MPA: MAP2407c(fadA3) MAP2436c(fadA4)-   MAV: MAV_(—)1544 MAV_(—)1573 MAV_(—)1863 MAV_(—)5081-   MSM: MSMEG_(—)2224 MSMEG_(—)4920-   MUL: MUL_(—)0357-   MVA: Mvan_(—)1976 Mvan_(—)1988 Mvan_(—)4305 Mvan_(—)4677    Mvan_(—)4891-   MGI: Mflv_(—)1347 Mflv_(—)1484 Mflv_(—)2040 Mflv_(—)2340    Mflv_(—)4356 Mflv_(—)4368-   MMC: Mmcs_(—)1758 Mmcs_(—)1769 Mmcs_(—)3796 Mmcs_(—)3864-   MKM: Mkms_(—)0251 Mkms_(—)1540 Mkms_(—)1805 Mkms_(—)1816    Mkms_(—)2836 Mkms_(—)3159

Mkms_(—)3286 Mkms_(—)3869 Mkms_(—)3938 Mkms_(—)4227 Mkms_(—)4411Mkms_(—)4580

Mkms_(—)4724 Mkms_(—)4764 Mkms_(—)4776

-   MJL: Mjls_(—)0231 Mjls_(—)1739 Mjls_(—)1750 Mjls_(—)2819    Mjls_(—)3119 Mjls_(—)3235

Mjls_(—)3800 Mjls_(—)3850 Mjls_(—)4110 Mjls_(—)4383 Mjls_(—)4705Mjls_(—)4876

Mjls_(—)5018 Mjls_(—)5063 Mjls_(—)5075

-   CGL: NCgl2309(cg12392)-   CGB: cg2625(pcaF)-   CEF: CE0731 CE2295-   CJK: jk1543(fadA3)-   NFA: nfa10750(fadA4)-   RHA: RHA1_ro01455 RHA1_ro01623 RHA1_ro01876 RHA1_ro02517(catF)

RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 RHA1_ro03892

RHA1_ro04599 RHA1_ro05257 RHA1_ro08871

-   SCO: SCO5399(SC8F4.03)-   SMA: SAV1384(fadA5) SAV2856(fadA1)-   ART: Arth_(—)1160 Arth_(—)2986 Arth_(—)3268 Arth_(—)4073-   NCA: Noca_(—)1371 Noca_(—)1797 Noca_(—)1828 Noca_(—)2764    Noca_(—)4142-   TFU: Tfu_(—)1520 Tfu_(—)2394-   FRA: Francci3_(—)3687-   FRE: Franean1_(—)1044 Franean1_(—)2711 Franean1_(—)2726    Franean1_(—)3929

Franean1_(—)4037 Franean1_(—)4577

-   FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB)-   ACE: Acel_(—)0626 Acel_(—)0672-   SEN: SACE_(—)1192(mmgA) SACE_(—)2736(fadA6) SACE_(—)4011(catF)

SACE_(—)6236(fadA4)

-   STP: Strop_(—)3610-   SAQ: Sare_(—)1316 Sare_(—)3991-   RXY: Rxyl_(—)1582 Rxyl_(—)1842 Rxyl_(—)2389 Rxyl_(—)2530-   FNU: FN0495-   BGA: BG0110(fadA)-   BAF: BAPKO_(—)0110(fadA)-   LIL: LA0457(thiL1) LA0828(thiL2) LA4139(fadA)-   LIC: LIC10396(phbA)-   LBJ: LBJ_(—)2862(paaJ-4)-   LBL: LBL_(—)0209(paaJ-4)-   SYN: slr1993(phaA)-   SRU: SRU_(—)1211(atoB) SRU_(—)1547-   CHU: CHU_(—)1910(atoB)-   GFO: GFO_(—)1507(atoB)-   FJO: Fjoh_(—)4612-   FPS: FP0770 FP1586 FP1725-   RRS: RoseRS 3911 RoseRS 4348-   RCA: Rcas_(—)0702 Rcas_(—)3206-   HAU: Haur_(—)0522-   DRA: DR_(—)1072 DR_(—)1428 DR_(—)1960 DR_(—)2480 DR_A0053-   DGE: Dgeo_(—)0755 Dgeo_(—)1305 Dgeo_(—)1441 Dgeo_(—)1883-   TTH: TTC0191 TTC0330-   TTJ: TTHA0559-   TME: Tme1_(—)1134-   FNO: Fnod_(—)0314-   PMO: Pmob_(—)0515-   HMA: rrnAC0896(acaB3) rrnAC2815(aca2) rrnAC3497(yqeF)

rrnB0240(aca1) rrnB0242(acaB2) rrnB0309(acaB1)

-   TAC: Ta0582-   TVO: TVN0649-   PTO: PT01505-   APE: APE_(—)2108-   SSO: SSO2377(acaB-4)-   STO: ST0514-   SAI: Saci_(—)0963 Saci_(—)1361(acaB1)-   MSE: Msed_(—)0656-   PAI: PAE1220-   PIS: Pisl_(—)0029 Pisl_(—)1301-   PCL: Pcal_(—)0781-   PAS: Pars_(—)0309 Pars_(—)1071-   CMA: Cmaq_(—)1941    Exemplary HMG-CoA Synthase Nucleic Acids and Polypeptides-   HSA: 3157(HMGCS1) 3158(HMGCS2)-   PTR: 457169(HMGCS2) 461892(HMGCS1)-   MCC: 702553(HMGCS1) 713541(HMGCS2)-   MMU: 15360(Hmgcs2) 208715(Hmgcs1)-   RNO: 24450(Hmgcs2) 29637(Hmgcs1)-   CFA: 479344(HMGCS1) 607923(HMGCS2)-   BTA: 407767(HMGCS1)-   SSC: 397673(CH242-38B5.1)-   GGA: 396379(HMGCS1)-   XLA: 380091(hmgcs1) 447204(MGC80816)-   DRE: 394060(hmgcs 1)-   SPU: 578259(L00578259)-   DME: Dmel_CG4311(Hmgs)-   CEL: F25B4.6-   ATH: AT4G11820(BAP1)-   OSA: 4331418 4347614-   CME: CMM189C-   SCE: YML126C(ERG13)-   AGO: AGOS_ADL356C-   PIC: PICST_(—)83020-   CAL: CaO19_(—)7312(Ca019.7312)-   CGR: CAGL0H04081g-   SPO: SPAC4F8.14c(hcs)-   MGR: MGG_(—)01026-   ANI: AN4923.2-   AFM: AFUA_(—)3G10660 AFUA_(—)8G07210-   AOR: AO090003000611 AO090010000487-   CNE: CNC05080 CNG02670-   UMA: UM05362.1-   ECU: ECU10_(—)0510-   DDI: DDBDRAFT_(—)0217522 DDB_(—)0219924(hgsA)-   TET: TTHERM_(—)00691190-   TBR: Tb927.8.6110-   YPE: YPO1457-   YPK: y2712(pksG)-   YPM: YP_(—)1349(pksG)-   YPA: YPA_(—)0750-   YPN: YPN_(—)2521-   YPP: YPDSF_(—)1517-   YPS: YPTB1475-   CBD: COXBU7E912_(—)1931-   TCX: Tcr_(—)1719-   DNO: DNO_(—)0799-   BMA: BMAA1212-   BPS: BPSS1002-   BPM: BURPS 1710b_A2613-   BPL: BURPS1106A_A1384-   BPD: BURPS668_A1470-   BTE: BTH_II1670-   MXA: MXAN_(—)3948(tac) MXAN_(—)4267(mvaS)-   BSU: BG10926(pksG)-   OIH: OB2248-   SAU: SA2334(mvaS)-   SAV: SAV2546(mvaS)-   SAM: MW2467(mvaS)-   SAR: SAR2626(mvaS)-   SAS: SAS2432-   SAC: SACOL2561-   SAB: SAB2420(mvaS)-   SAA: SAUSA300_(—)2484-   SAO: SAOUHSC_(—)02860-   SAJ: SaurJH9_(—)2569-   SAH: SaurJH1_(—)2622-   SEP: SE2110-   SER: SERP2122-   SHA: SH0508(mvaS)-   SSP: SSP0324-   LMO: lmo1415-   LMF: LMOf2365_(—)1434(mvaS)-   LIN: lin1454-   LWE: lwe1432(mvaS)-   LLA: L13187(hmcM)-   LLC: LACR_(—)1666-   LLM: llmg_(—)0929(hmcM)-   SPY: SPy_(—)0881(mvaS.2)-   SPZ: M5005_Spy_(—)0687(mvaS.1)-   SPM: spyM18_(—)0942(mvaS2)-   SPG: SpyM3_(—)0600(mvaS.2)-   SPS: SPs1253-   SPH: MGAS10270_Spy0745(mvaS1)-   SPI: MGAS10750_Spy0779(mvaS1)-   SPJ: MGAS2096_Spy0759(mvaS1)-   SPK: MGAS9429_Spy0743(mvaS1)-   SPF: SpyM51121(mvaS)-   SPA: M6_Spy0704-   SPB: M28_Spy0667(mvaS.1)-   SPN: SP 1727-   SPR: spr1571(mvaS)-   SPD: SPD_(—)1537(mvaS)-   SAG: SAG1316-   SAN: gbs1386-   SAK: SAK_(—)1347-   SMU: SMU.943c-   STC: str0577(mvaS)-   STL: stu0577(mvaS)-   STE: STER_(—)0621-   SSA: SSA_(—)0338(mvaS)-   SSU: SSU05_(—)1641-   SSV: SSU98_(—)1652-   SGO: SGO_(—)0244-   LPL: lp_(—)2067(mvaS)-   LJO: LJ1607-   LAC: LBA0628(hmcS)-   LSA: LSA1484(mvaS)-   LSL: LSL_(—)0526-   LDB: Ldb0881(mvaS)-   LBU: LBUL_(—)0806-   LBR: LVIS_(—)1363-   LCA: LSEI_(—)1785-   LGA: LGAS_(—)1372-   LRE: Lreu_(—)0676-   PPE: PEPE_(—)0868-   EFA: EF1363-   OOE: OEOE_(—)0968-   LME: LEUM_(—)1184-   NFA: nfa22120-   SEN: SACE_(—)4570(pksG)-   BBU: BB0683-   BGA: BG0706-   BAF: BAPKO_(—)0727-   FJO: Fjoh_(—)0678-   HAL: VNG1615G(mvaB)-   HMA: rrnAC1740(mvaS)-   HWA: HQ2868A(mvaB)-   NPH: NP2608A(mvaB_(—)1) NP4836A(mvaB_(—)2)    Exemplary Hydroxymethylglutaryl-CoA Reductase Nucleic Acids and    Polypeptides-   HSA: 3156(HMGCR)-   PTR: 471516(HMGCR)-   MCC: 705479(HMGCR)-   MMU: 15357(Hmgcr)-   RNO: 25675(Hmgcr)-   CFA: 479182(HMGCR)-   BTA: 407159(HMGCR)-   GGA: 395145(RCJMB04_(—)14m24)-   SPU: 373355(LOC373355)-   DME: Dmel_CG10367(Hmgcr)-   CEL: F08F8.2-   OSA: 4347443-   SCE: YLR450W(HMG2) YML075C(HMG1)-   AGO: AGOS_AER152W-   CGR: CAGL0L11506g-   SPO: SPCC162.09c(hmg1)-   ANI: AN3817.2-   AFM: AFUA_(—)1G11230 AFUA_(—)2G03700-   AOR: AO090103000311 AO090120000217-   CNE: CNF04830-   UMA: UM03014.1-   ECU: ECU10_(—)1720-   DDI: DDB_(—)0191125(hmgA) DDB_(—)0215357(hmgB)-   TBR: Tb927.6.4540-   TCR: 506831.40 509167.20-   LMA: LmjF30.3190-   VCH: VCA0723-   VCO: VCO395_(—)0662-   VVU: VV2_(—)0117-   VVY: VVA0625-   VPA: VPA0968-   VFI: VFA0841-   PAT: Patl_(—)0427-   CBU: CBU_(—)0030 CBU_(—)0610-   CBD: COXBU7E912_(—)0151 COXBU7E912_(—)0622(hmgA)-   TCX: Tcr_(—)1717-   DNO: DNO_(—)0797-   CVI: CV_(—)1806-   SUS: Acid_(—)5728 Acid_(—)6132-   SAU: SA2333(mvaA)-   SAV: SAV2545(mvaA)-   SAM: MW2466(mvaA)-   SAB: SAB2419c(mvaA)-   SEP: SE2109-   LWE: lwe0819(mvaA)-   LLA: L10433(mvaA)-   LLC: LACR_(—)1664-   LLM: llmg_(—)0931(mvaA)-   SPY: SPy_(—)0880(mvaS.1)-   SPM: spyM18_(—)0941(mvaS1)-   SPG: SpyM3_(—)0599(mvaS.1)-   SPS: SPs1254-   SPH: MGAS 10270_Spy0744-   SPI: MGAS 10750_Spy0778-   SPJ: MGAS2096_Spy0758-   SPK: MGAS9429_Spy0742-   SPA: M6_Spy0703-   SPN: SP_(—)1726-   SAG: SAG1317-   SAN: gbs1387-   STC: str0576(mvaA)-   STL: stu0576(mvaA)-   STE: STER_(—)0620-   SSA: SSA_(—)0337(mvaA)-   LPL: lp_(—)0447(mvaA)-   LJO: LJ1608-   LS L: LSL_(—)0224-   LBR: LVIS_(—)0450-   LGA: LGAS_(—)1373-   EFA: EF1364-   NFA: nfa22110-   BGA: BG0708(mvaA)-   SRU: SRU_(—)2422-   FPS: FP2341-   MMP: MMP0087(hmgA)-   MMQ: MmarC5_(—)1589-   MAC: MA3073(hmgA)-   MBA: Mbar_A1972-   MMA: MM_(—)0335-   MBU: Mbur_(—)1098-   MHU: Mhun_(—)3004-   MEM: Memar_(—)2365-   MBN: Mboo_(—)0137-   MTH: MTH562-   MST: Msp_(—)0584(hmgA)-   MSI: Msm_(—)0227-   MKA: MK0355(HMG1)-   AFU: AF1736(mvaA)-   HAL: VNG1875G(mvaA)-   HMA: rrnAC3412(mvaA)-   HWA: HQ3215A(hmgR)-   NPH: NP0368A(mvaA_(—)2) NP2422A(mvaA_(—)1)-   TAC: Ta0406m-   TVO: TVN1168-   PTO: PTO1143-   PAB: PAB2106(mvaA)-   PFU: PF1848-   TKO: TK0914-   RCI: RCIX1027(hmgA) RCIX376(hmgA)-   APE: APE_(—)1869-   IHO: Igni_(—)0476-   HBU: Hbut_(—)1531-   SSO: SSO0531-   STO: ST1352-   SAI: Saci_(—)1359-   PAI: PAE2182-   PIS: Pisl_(—)0814-   PCL: Pcal_(—)1085-   PAS: Pars_(—)0796    Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides-   HSA: 4598(MVK)-   MCC: 707645(MVK)-   MMU: 17855(Mvk)-   RNO: 81727(Mvk)-   CFA: 486309(MVK)-   BTA: 505792(MVK)-   GGA: 768555(MVK)-   DRE: 492477(zgc:103473)-   SPU: 585785(LOC585785)-   DME: Dmel_CG33671-   OSA: 4348331-   SCE: YMR208W(ERG12)-   AGO: AGOS_AER335W-   PIC: PICST_(—)40742(ERG12)-   CGR: CAGL0F03861g-   SPO: SPAC13G6.11c-   MGR: MGG_(—)06946-   ANI: AN3869.2-   AFM: AFUA_(—)4G07780-   AOR: AO090023000793-   CNE: CNK01740-   ECU: ECU09_(—)1780-   DDI: DDBDRAFT_(—)0168621-   TET: TTHERM_(—)00637680-   TBR: Tb927.4.4070-   TCR: 436521.9 509237.10-   LMA: LmjF31.0560-   CBU: CBU_(—)0608 CBU_(—)0609-   CBD: COXBU7E912_(—)0620(mvk)-   LPN: lpg2039-   LPF: lpl2017-   LPP: lpp2022-   BBA: Bd1027(lmbP) Bd1630(mvk)-   MXA: MXAN_(—)5019(mvk)-   OIH: OB0225-   SAU: SA0547(mvaK1)-   SAV: SAV0590(mvaK1)-   SAM: MW0545(mvaK1)-   SAR: SAR0596(mvaK1)-   SAS: SAS0549-   SAC: SACOL0636(mvk)-   SAB: SAB0540(mvaK1)-   SAA: SAUSA300_(—)0572(mvk)-   SAO: SAOUHSC_(—)00577-   SEP: SE0361-   SER: SERP0238(mvk)-   SHA: SH2402(mvaK1)-   SSP: SSP2122-   LMO: lmo0010-   LMF: LMOf2365_(—)0011-   LIN: lin0010-   LWE: lwe0011(mvk)-   LLA: L7866(yeaG)-   LLC: LACR_(—)0454-   LLM: llmg_(—)0425(mvk)-   SPY: SPy_(—)0876(mvaK1)-   SPZ: M5005_Spy_(—)0682(mvaK1)-   SPM: spyM18_(—)0937(mvaK1)-   SPG: SpyM3_(—)0595(mvaK1)-   SPS: SPs1258-   SPH: MGAS10270_Spy0740(mvaK1)-   SPI: MGAS10750_Spy0774(mvaK1)-   SPJ: MGAS2096_Spy0753(mvaK1)-   SPK: MGAS9429_Spy0737(mvaK1)-   SPF: SpyM51126(mvaK1)-   SPA: M6_Spy0699-   SPB: M28_Spy0662(mvaK1)-   SPN: SP_(—)0381-   SPR: spr0338(mvk)-   SPD: SPD_(—)0346(mvk)-   SAG: SAG1326-   SAN: gbs1396-   SAK: SAK_(—)1357(mvk)-   SMU: SMU.181-   STC: str0559(mvaK1)-   STL: stu0559(mvaK1)-   STE: STER_(—)0598-   SSA: SSA_(—)0333(mvaK1)-   SSU: SSU05_(—)0289-   SSV: SSU98_(—)0285-   SGO: SGO_(—)0239(mvk)-   LPL: lp_(—)1735(mvaK1)-   LJO: LJ1205-   LAC: LBA1167(mvaK)-   LSA: LSA0908(mvaK1)-   LSL: LSL_(—)0685(eRG)-   LDB: Ldb0999(mvk)-   LBU: LBUL_(—)0906-   LBR: LVIS_(—)0858-   LCA: LSEI_(—)1491-   LGA: LGAS_(—)1033-   LRE: Lreu_(—)0915-   PPE: PEPE_(—)0927-   EFA: EF0904(mvk)-   OOE: OEOE_(—)1100-   LME: LEUM_(—)1385-   NFA: nfa22070-   BGA: BG0711-   BAF: BAPKO_(—)0732-   FPS: FP0313-   MMP: MMP1335-   MAE: Maeo_(—)0775-   MAC: MA0602(mvk)-   MBA: Mbar_A1421-   MMA: MM_(—)1762-   MBU: Mbur_(—)2395-   MHU: Mhun_(—)2890-   MEM: Memar_(—)1812-   MBN: Mboo_(—)2213-   MST: Msp_(—)0858(mvk)-   MSI: Msm_(—)1439-   MKA: MK0993(ERG12)-   HAL: VNG1145G(mvk)-   HMA: rrnAC0077(mvk)-   HWA: HQ2925A(mvk)-   NPH: NP2850A(mvk)-   PTO: PTO1352-   PHO: PH1625-   PAB: PAB0372(mvk)-   PFU: PF1637(mvk)-   TKO: TK1474-   RCI: LRC399(mvk)-   APE: APE_(—)2439-   HBU: Hbut_(—)0877-   SSO: SSO0383-   STO: ST2185-   SAI: Saci_(—)2365(mvk)-   MSE: Msed_(—)1602-   PAI: PAE3108-   PIS: Pisl_(—)0467-   PCL: Pcal_(—)1835    Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides Homologus    to Methanosarcina mazei Mevalonate Kinase-   NP_(—)633786.1 mevalonate kinase Methanosarcina mazei Go1-   YP_(—)304960.1 mevalonate kinase Methanosarcina barkeri str. Fusaro-   NP_(—)615566.1 mevalonate kinase Methanosarcina acetivorans C2A-   YP_(—)566996.1 mevalonate kinase Methanococcoides burtonii DSM 6242-   YP_(—)684687.1 mevalonate kinase uncultured methanogenic archaeon    RC-1-   YP_(—)183887.1 mevalonate kinase Thermococcus kodakarensis KOD1-   NP_(—)126232.1 mevalonate kinase Pyrococcus abyssi GE5-   NP_(—)143478.1 mevalonate kinase Pyrococcus horikoshii OT3-   NP_(—)579366.1 mevalonate kinase Pyrococcus furiosus DSM 3638-   YP_(—)842907.1 mevalonate kinase Methanosaeta thermophila PT-   YP_(—)327075.1 mevalonate kinase Natronomonas pharaonis DSM 2160-   YP_(—)658630.1 mevalonate kinase Haloquadratum walsbyi DSM 16790-   YP_(—)134862.1 mevalonate kinase Haloarcula marismortui ATCC 43049-   YP_(—)001405370.1 mevalonate kinase Candidatus Methanoregula boonei    6A8-   YP_(—)001030120.1 mevalonate kinase Methanocorpusculum labreanum Z-   YP_(—)447890.1 putative mevalonate kinase Methanosphaera stadtmanae    DSM 3091-   YP_(—)920295.1 mevalonate kinase Thermofilum pendens Hrk 5-   ZP_(—)02015315.1 mevalonate kinase Halorubrum lacusprofundi ATCC    49239-   NP_(—)280049.1 mevalonate kinase Halobacterium sp. NRC-1-   YP_(—)001274012.1 mevalonate kinase Methanobrevibacter smithii ATCC    35061-   YP_(—)001435347.1 mevalonate kinase Ignicoccus hospitalis KIN4/I-   YP_(—)001540788.1 mevalonate kinase Caldivirga maquilingensis IC-167-   Q50559 KIME_METTH mevalonate kinase (MK)-   NP_(—)275189.1 mevalonate kinase Methanothermobacter    thermautotrophicus str.-   NP_(—)071114.1 mevalonate kinase (mvk) Archaeoglobus fulgidus DSM    4304-   YP_(—)504301.1 mevalonate kinase Methanospirillum hungatei JF-1-   YP_(—)001040239.1 mevalonate kinase Staphylothermus marinus F1-   YP_(—)001047720.1 mevalonate kinase Methanoculleus marisnigri JR1-   NP_(—)614276.1 mevalonate kinase Methanopyrus kandleri AV19-   YP_(—)001737496.1 mevalonate kinase Candidatus Korarchaeum    cryptofilum OPF8-   YP_(—)256937.1 mevalonate kinase Sulfolobus acidocaldarius DSM 639-   NP_(—)341921.1 mevalonate kinase Sulfolobus solfataricus P2-   YP_(—)001276466.1 mevalonate kinase Roseiflexus sp. RS-1-   YP_(—)001581649.1 mevalonate kinase Nitrosopumilus maritimus SCM1-   NP_(—)378182.1 hypothetical protein ST2185 Sulfolobus tokodaii str.    7-   YP_(—)001547075.1 mevalonate kinase Herpetosiphon aurantiacus ATCC    23779-   YP_(—)001056718.1 mevalonate kinase Pyrobaculum calidifontis JCM    11548-   YP_(—)001431846.1 mevalonate kinase Roseiflexus castenholzii DSM    13941-   YP_(—)001153805.1 mevalonate kinase Pyrobaculum arsenaticum DSM    13514-   AAG02440.1AF290093_(—)1 mevalonate kinase Enterococcus faecalis-   NP_(—)814642.1 mevalonate kinase Enterococcus faecalis V583-   YP_(—)001634502.1 mevalonate kinase Chloroflexus aurantiacus J-10-fl-   XP_(—)790690.1 similar to Mevalonate kinase (MK) Strongylocentrotus    purpuratus-   NP_(—)560495.1 mevalonate kinase Pyrobaculum aerophilum str. IM2-   YP_(—)929988.1 mevalonate kinase Pyrobaculum islandicum DSM 4184-   ZP_(—)01465063.1 mevalonate kinase Stigmatella aurantiaca DW4/3-1-   ZP_(—)01906658.1 mevalonate kinase Plesiocystis pacifica SIR-1-   NP_(—)248080.1 mevalonate kinase Methanocaldococcus jannaschii DSM    2661-   1KKHA chain A of the Methanococcus jannaschii mevalonate kinase    Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides Homologus    to Lactobacillus sakei Mevalonate Kinase

YP_(—)395519.1 mevalonate kinase Lactobacillus sakei subsp. sakei 23K

-   YP_(—)535578.1 mevalonate kinase Lactobacillus salivarius UCC118-   YP_(—)804427.1 mevalonate kinase Pediococcus pentosaceus ATCC 25745-   YP_(—)001271514.1 mevalonate kinase Lactobacillus reuteri F275-   ZP_(—)03073995.1 mevalonate kinase Lactobacillus reuteri 100-23-   YP_(—)795031.1 mevalonate kinase Lactobacillus brevis ATCC 367-   ZP_(—)02185318.1 mevalonate kinase Carnobacterium sp. AT7-   YP_(—)001844008.1 mevalonate kinase Lactobacillus fermentum IFO 3956-   NP_(—)266560.1 mevalonate kinase Lactococcus lactis subsp. lactis    Il1403-   YP_(—)818851.1 mevalonate kinase Leuconostoc mesenteroides subsp.    mesenteroides ATCC 8293-   NP_(—)785308.1 mevalonate kinase Lactobacillus plantarum WCFS1-   ZP_(—)00604007.1 Mevalonate kinase Enterococcus faecium DO-   YP_(—)808480.1 mevalonate kinase Lactococcus lactis subsp. cremoris    SK11-   YP_(—)001031775.1 mevalonate kinase Lactococcus lactis subsp.    cremoris MG1363-   NP_(—)814642.1 mevalonate kinase Enterococcus faecalis V583-   AAG02440.1 AF290093_(—)1 mevalonate kinase Enterococcus faecalis    Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides Homologus    to Streptomyces sp. CL190 Mevalonate Kinase-   BAB07790.1 mevalonate kinase Streptomyces sp. CL190-   BAD86800.1 mevalonate kinase Streptomyces sp. KO-3988-   BAB07817.1 mevalonate kinase Kitasatospora griseola-   ABS50475.1 NapT6 Streptomyces sp. CNQ525-   ABS50448.1 NapT6 Streptomyces aculeolatus-   BAE78977.1 mevalonate kinase Streptomyces sp. KO-3988-   CAL34097.1 putative mevalonate kinase Streptomyces cinnamonensis-   BAD07375.1 mevalonate kinase Actinoplanes sp. A40644-   YP_(—)118418.1 putative mevalonate kinase Nocardia farcinica IFM    10152-   YP_(—)818851.1 mevalonate kinase Leuconostoc mesenteroides subsp.    mesenteroides ATCC 8293-   YP_(—)001620791.1 mevalonate kinase Acholeplasma laidlawii PG-8A-   NP_(—)720650.1 putative mevalonate kinase Streptococcus mutans UA159-   YP_(—)001031775.1 mevalonate kinase Lactococcus lactis subsp.    cremoris MG1363-   ZP_(—)02689018.1 mevalonate kinase Listeria monocytogenes FSL J2-071-   NP_(—)266560.1 mevalonate kinase Lactococcus lactis subsp. lactis    Il1403-   YP_(—)395519.1 mevalonate kinase Lactobacillus sakei subsp. sakei    23K-   YP_(—)808480.1 mevalonate kinase Lactococcus lactis subsp. cremoris    SK11-   ZP_(—)01926008.1 mevalonate kinase Listeria monocytogenes FSL N1-017-   ZP_(—)01942559.1 mevalonate kinase Listeria monocytogenes HPB2262-   YP_(—)012624.1 mevalonate kinase Listeria monocytogenes str. 4b    F2365-   YP_(—)001727922.1 mevalonate kinase Leuconostoc citreum KM20-   NP_(—)469357.1 hypothetical protein lin0010 Listeria innocua    Clip11262-   ZP_(—)00875673.1 Mevalonate kinase Streptococcus suis 89/1591-   ZP_(—)00604007.1 Mevalonate kinase Enterococcus faecium DO-   ZP_(—)00230799.1 mevalonate kinase Listeria monocytogenes str. 4b    H7858-   YP_(—)139080.1 mevalonate kinase Streptococcus thermophilus LMG    18311-   YP_(—)140970.1 mevalonate kinase Streptococcus thermophilus CNRZ1066-   ZP_(—)01544345.1 mevalonate kinase Oenococcus oeni ATCC BAA-1163-   YP_(—)001197657.1 mevalonate kinase Streptococcus suis 05ZYH33-   YP_(—)810664.1 mevalonate kinase Oenococcus oeni PSU-1-   NP_(—)463543.1 hypothetical protein lmo0010 Listeria monocytogenes    EGD-e-   YP_(—)848214.1 mevalonate kinase Listeria welshimeri serovar 6b str.    SLCC5334-   ZP_(—)01695505.1 mevalonate kinase Bacillus coagulans 36D1-   YP_(—)804427.1 mevalonate kinase Pediococcus pentosaceus ATCC 25745-   YP_(—)820062.1 mevalonate kinase Streptococcus thermophilus LMD-9-   NP_(—)814642.1 mevalonate kinase Enterococcus faecalis V583-   AAG02440.1 AF290093_(—)1 mevalonate kinase Enterococcus faecalis-   YP_(—)598349.1 mevalonate kinase Streptococcus pyogenes MGAS10270-   YP_(—)535578.1 mevalonate kinase Lactobacillus salivarius UCC118-   YP_(—)001851498.1 mevalonate kinase, Erg12 Mycobacterium marinum M-   ZP_(—)01817104.1 mevalonate kinase Streptococcus pneumoniae SP3-BS71-   YP_(—)002037061.1 mevalonate kinase Streptococcus pneumoniae G54-   NP_(—)357932.1 mevalonate kinase Streptococcus pneumoniae R6-   ZP_(—)02710031.1 mevalonate kinase Streptococcus pneumoniae    CDC1087-00-   NP_(—)344908.1 mevalonate kinase Streptococcus pneumoniae TIGR4-   YP_(—)001547075.1 mevalonate kinase Herpetosiphon aurantiacus ATCC    23779-   AAG02455.1 AF290099_(—)1 mevalonate kinase Streptococcus pneumoniae-   ZP_(—)01819603.1 mevalonate kinase Streptococcus pneumoniae SP6-BS73-   YP_(—)001271514.1 mevalonate kinase Lactobacillus reuteri F275-   NP_(—)965060.1 mevalonate kinase Lactobacillus johnsonii NCC 533-   ZP_(—)02919501.1 hypothetical protein STRINF_(—)00343 Streptococcus    infantarius-   YP_(—)001034340.1 mevalonate kinase, putative Streptococcus    sanguinis SK36-   YP_(—)001844008.1 mevalonate kinase Lactobacillus fermentum IFO 3956-   ZP_(—)03073995.1 mevalonate kinase Lactobacillus reuteri 100-23-   NP_(—)688324.1 mevalonate kinase, putative Streptococcus agalactiae    2603V/R-   YP_(—)907150.1 mevalonate kinase, Erg12 Mycobacterium ulcerans Agy99-   NP_(—)691146.1 mevalonate kinase Oceanobacillus iheyensis HTE831-   YP_(—)795031.1 mevalonate kinase Lactobacillus brevis ATCC 367-   YP_(—)002123449.1 mevalonate kinase Mvk Streptococcus equi subsp.    zooepidemicus str. MGCS10565-   YP_(—)001449558.1 mevalonate kinase Streptococcus gordonii str.    Challis substr. CH1-   ZP_(—)02185318.1 mevalonate kinase Carnobacterium sp. AT7-   YP_(—)001634502.1 mevalonate kinase Chloroflexus aurantiacus J-10-fl-   YP_(—)812921.1 mevalonate kinase Lactobacillus delbrueckii subsp.    bulgaricus ATCC BAA-365-   YP_(—)814846.1 mevalonate kinase Lactobacillus gasseri ATCC 33323-   YP_(—)001987652.1 Mevalonate kinase Lactobacillus casei-   YP_(—)618979.1 mevalonate kinase Lactobacillus delbrueckii subsp.    bulgaricus ATCC 11842-   NP_(—)664399.1 mevalonate kinase Streptococcus pyogenes MGAS315-   YP_(—)806709.1 mevalonate kinase Lactobacillus casei ATCC 334-   YP_(—)060017.1 mevalonate kinase Streptococcus pyogenes MGAS10394-   YP_(—)280130.1 mevalonate kinase Streptococcus pyogenes MGAS6180-   NP_(—)269075.1 mevalonate kinase Streptococcus pyogenes M1 GAS-   YP_(—)001276466.1 mevalonate kinase Roseiflexus sp. RS-1-   NP_(—)607080.1 mevalonate kinase Streptococcus pyogenes MGAS8232-   NP_(—)785308.1 mevalonate kinase Lactobacillus plantarum WCFS1-   ABH11598.1 GMP synthase, mevalonate kinase Lactobacillus helveticus    CNRZ32-   YP_(—)001577580.1 mevalonate kinase Lactobacillus helveticus DPC    4571-   YP_(—)001431846.1 mevalonate kinase Roseiflexus castenholzii DSM    13941-   YP_(—)302212.1 mevalonate kinase Staphylococcus saprophyticus subsp.    saprophyticus ATCC 15305-   YP_(—)040044.1 mevalonate kinase Staphylococcus aureus subsp. aureus    MRSA252-   AAG02424.1 AF290087_(—)1 mevalonate kinase Staphylococcus aureus-   NP_(—)645362.1 mevalonate kinase Staphylococcus aureus subsp. aureus    MW2-   ZP_(—)01514039.1 mevalonate kinase Chloroflexus aggregans DSM 9485-   YP_(—)194037.1 mevalonate kinase Lactobacillus acidophilus NCFM-   YP_(—)254317.1 mevalonate kinase Staphylococcus haemolyticus    JCSC1435-   YP_(—)187834.1 mevalonate kinase Staphylococcus epidermidis RP62A-   AAG02435.1 AF290091_(—)1 mevalonate kinase Staphylococcus    epidermidis-   YP_(—)183887.1 mevalonate kinase Thermococcus kodakarensis KOD1-   NP_(—)143478.1 mevalonate kinase Pyrococcus horikoshii OT3-   ZP_(—)00780842.1 mevalonate kinase Streptococcus agalactiae 18RS21-   NP_(—)579366.1 mevalonate kinase Pyrococcus furiosus DSM 3638-   NP_(—)126232.1 mevalonate kinase Pyrococcus abyssi GE5-   NP_(—)371114.1 mevalonate kinase Staphylococcus aureus subsp. aureus    Mu50-   YP_(—)001040239.1 mevalonate kinase Staphylothermus marinus F1-   NP_(—)763916.1 mevalonate kinase Staphylococcus epidermidis ATCC    12228-   YP_(—)633174.1 mevalonate kinase Myxococcus xanthus DK 1622-   YP_(—)920295.1 mevalonate kinase Thermofilum pendens Hrk 5-   NP_(—)148611.1 mevalonate kinase Aeropyrum pernix K1-   NP_(—)633786.1 mevalonate kinase Methanosarcina mazei Go1    Exemplary Phosphomevalonate Kinase Nucleic Acids and Polypeptides-   HSA: 10654(PMVK)-   PTR: 457350(PMVK)-   MCC: 717014(PMVK)-   MMU: 68603(Pmvk)-   CFA: 612251(PMVK)-   BTA: 513533(PMVK)-   DME: Dmel_CG10268-   ATH: AT1G31910-   OSA: 4332275-   SCE: YMR220W(ERG8)-   AGO: AGOS_AER354W-   PIC: PICST_(—)52257(ERG8)-   CGR: CAGLOF03993g-   SPO: SPAC343.01c-   MGR: MGG_(—)05812-   ANI: AN2311.2-   AFM: AFUA_(—)5G10680-   AOR: AO090010000471-   CNE: CNM00100-   UMA: UM00760.1-   DDI: DDBDRAFT_(—)0184512-   TBR: Tb09.160.3690-   TCR: 507913.20 508277.140-   LMA: LmjF15.1460-   MXA: MXAN_(—)5017-   OIH: OB0227-   SAU: SA0549(mvaK2)-   SAV: SAV0592(mvaK2)-   SAM: MW0547(mvaK2)-   SAR: SAR0598(mvaK2)-   SAS: SAS0551-   SAC: SACOL0638-   SAB: SAB0542(mvaK2)-   SAA: SAUSA300_(—)0574-   SAO: SAOUHSC_(—)00579-   SAT: SaurJII9_(—)0615-   SEP: SE0363-   SER: SERP0240-   SHA: SH2400(mvaK2)-   SSP: SSP2120-   LMO: lmo0012-   LMF: LMOf2365_(—)0013-   LIN: lin0012-   LWE: lwe0013-   LLA: L10014(yebA)-   LLC: LACR_(—)0456-   LLM: llmg_(—)0427-   SPY: SPy_(—)0878(mvaK2)-   SPZ: M5005_Spy_(—)0684(mvaK2)-   SPM: spyM18_(—)0939-   SPG: SpyM3_(—)0597(mvaK2)-   SPS: SPs1256-   SPH: MGAS10270_Spy0742(mvaK2)-   SPI: MGAS10750_Spy0776(mvaK2)-   SPJ: MGAS2096_Spy0755(mvaK2)-   SPK: MGAS9429_Spy0739(mvaK2)-   SPF: SpyM51124(mvaK2)-   SPA: M6_Spy0701-   SPB: M28_Spy0664(mvaK2)-   SPN: SP_(—)0383-   SPR: spr0340(mvaK2)-   SPD: SPD_(—)0348(mvaK2)-   SAG: SAG1324-   SAN: gbs1394-   SAK: SAK_(—)1355-   SMU: SMU.938-   STC: str0561(mvaK2)-   STL: stu0561(mvaK2)-   STE: STER_(—)0600-   SSA: SSA_(—)0335(mvaK2)-   SSU: SSU05_(—)0291-   SSV: SSU98_(—)0287-   SGO: SGO_(—)0241-   LPL: lp_(—)1733(mvaK2)-   LJO: LJ1207-   LAC: LBA1169-   LSA: LSA0906(mvaK2)-   LSL: LSL_(—)0683-   LDB: Ldb0997(mvaK)-   LBU: LBUL_(—)0904-   LBR: LVIS_(—)0860-   LCA: LSEI_(—)1092-   LGA: LGAS_(—)1035-   LRE: Lreu_(—)0913-   PPE: PEPE_(—)0925-   EFA: EF0902-   NFA: nfa22090-   BGA: BG0710-   BAF: BAPKO_(—)0731-   NPH: NP2852A-   SSO: SSO2988-   STO: ST0978-   SAL Saci_(—)1244    Exemplary Diphosphomevalonate Decarboxylase Nucleic Acids and    Polypeptides-   HSA: 4597(MVD)-   PTR: 468069(MVD)-   MCC: 696865(MVD)-   MMU: 192156(Mvd)-   RNO: 81726(Mvd)-   CFA: 489663(MVD)-   GGA: 425359(MVD)-   DME: Dmel_CG8239-   SCE: YNR043W(MVD1)-   AGO: AGOS_AGL232C-   PIC: PICST_(—)90752-   CGR: CAGL0C03630g-   SPO: SPAC24C9.03-   MGR: MGG 09750-   ANI: AN4414.2-   AFM: AFUA_(—)4G07130-   AOR: AO090023000862-   CNE: CNL04950-   UMA: UM05179.1-   DDI: DDBDRAFT_(—)0218058-   TET: TTHERM_(—)00849200-   TBR: Tb10.05.0010 Tb10.61.2745-   TCR: 507993.330 511281.40-   LMA: LmjF18.0020-   CBU: CBU_(—)0607(mvaD)-   CBD: COXBU7E912_(—)0619(mvaD)-   LPN: lpg2040-   LPF: lpl2018-   LPP: lpp2023-   TCX: Tcr_(—)1734-   DNO: DNO_(—)0504(mvaD)-   BBA: Bd1629-   MXA: MXAN_(—)5018(mvaD)-   OIH: OB0226-   SAU: SA0548(mvaD)-   SAV: SAV0591(mvaD)-   SAM: MW0546(mvaD)-   SAR: SAR0597(mvaD)-   SAS: SAS0550-   SAC: SACOL0637(mvaD)-   SAB: SAB0541(mvaD)-   SAA: SAUSA300_(—)0573(mvaD)-   SAO: SAOUHSC_(—)00578-   SAJ: SaurJH9_(—)0614-   SAH: SaurJH1_(—0629)-   SEP: SE0362-   SER: SERP0239(mvaD)-   SHA: SH2401(mvaD)-   SSP: SSP2121-   LMO: lmo0011-   LMF: LMOf2365_(—)0012(mvaD)-   LIN: lin0011-   LWE: lwe0012(mvaD)-   LLA: L9089(yeaH)-   LLC: LACR_(—)0455-   LLM: llmg_(—)0426(mvaD)-   SPY: SPy_(—)0877(mvaD)-   SPZ: M5005_Spy_(—)0683(mvaD)-   SPM: spyM18_(—)0938(mvd)-   SPG: SpyM3_(—)0596(mvaD)-   SPS: SPs1257-   SPH: MGAS10270_Spy0741(mvaD)-   SPI: MGAS10750_Spy0775(mvaD)-   SPJ: MGAS2096_Spy0754(mvaD)-   SPK: MGAS9429_Spy0738(mvaD)-   SPF: SpyM51125(mvaD)-   SPA: M6_Spy0700-   SPB: M28_Spy0663(mvaD)-   SPN: SP_(—)0382-   SPR: spr0339(mvd1)-   SPD: SPD_(—)0347(mvaD)-   SAG: SAG1325(mvaD)-   SAN: gbs1395-   SAK: SAK_(—)1356(mvaD)-   SMU: SMU.937-   STC: str0560(mvaD)-   STL: stu0560(mvaD)-   STE: STER_(—)0599-   SSA: SSA_(—)0334(mvaD)-   SSU: SSU05_(—)0290-   SSV: SSU98_(—)0286-   SGO: SGO_(—)0240(mvaD)-   LPL: lp_(—)1734(mvaD)-   LJO: LJ1206-   LAC: LBA1168(mvaD)-   LSA: LSA0907(mvaD) LSL: LSL_(—)0684-   LDB: Ldb0998(mvaD)-   LBU: LBUL_(—)0905-   LBR: LVIS_(—)0859-   LCA: LSEI_(—)1492-   LGA: LGAS_(—)1034-   LRE: Lreu_(—)0914-   PPE: PEPE_(—)0926-   EFA: EF0903(mvaD)-   LME: LEUM_(—)1386-   NFA: nfa22080-   BBU: BB0686-   BGA: BG0709-   BAF: BAPKO_(—)0730-   GFO: GFO_(—)3632-   FPS: FP0310(mvaD)-   HAU: Haur_(—)1612-   HAL: VNG0593G(dmd)-   HMA: rrnAC1489(dmd)-   HWA: HQ1525A(mvaD)-   NPH: NP1580A(mvaD)-   PTO: PTO478 PTO1356-   SSO: SSO2989-   STO: ST0977-   SAI: Saci_(—)1245(mvd)-   MSE: Msed_(—)1576    Exemplary Isopentenyl Phosphate Kinases (IPK) Nucleic Acids and    Polypeptides-   Methanobacterium thermoautotrophicum gil2621082-   Methanococcus jannaschii DSM 2661 gil1590842;-   Methanocaldococcus jannaschii gil1590842-   Methanothermobacter thermautotrophicus gil2621082-   Picrophilus torridus DSM9790 (IG-57) gil48477569-   Pyrococcus abyssi gil14520758-   Pyrococcus horikoshii OT3 gil3258052-   Archaeoglobus fulgidus DSM4304 gil2648231    Exemplary Isopentenyl-Diphosphate Delta-Isomerase (IDI) Nucleic    Acids and Polypeptides-   HSA: 3422(IDI1) 91734(ID12)-   PTR: 450262(IDI2) 450263(IDI1)-   MCC: 710052(LOC710052) 721730(LOC721730)-   MMU: 319554(Idi1)-   RNO: 89784(Idi1)-   GGA: 420459(IDI1)-   XLA: 494671(LOC494671)-   XTR: 496783(idi2)-   SPU: 586184(LOC586184)-   CEL: K06H7.9(idi-1)-   ATH: AT3G02780(IPP2)-   OSA: 4338791 4343523-   CME: CMB062C-   SCE: YPL117C(IDI1)-   AGO: AGOS_ADL268C-   PIC: PICST_(—)68990(IDI1)-   CGR: CAGL0J06952g-   SPO: SPBC106.15(idi1)-   ANI: AN0579.2-   AFM: AFUA_(—)6G11160-   AOR: AO090023000500-   CNE: CNA02550-   UMA: UM04838.1-   ECU: ECUO2_(—)0230-   DDI: DDB_(—)0191342(ipi)-   TET: TTHERM_(—)00237280 TTHERM_(—)00438860-   TBR: Tb09.211.0700-   TCR: 408799.19 510431.10-   LMA: LmjF35.5330-   EHI: 46.t00025-   ECO: b2889(idi)-   ECJ: JW2857(idi)-   ECE: Z4227-   ECS: ECs3761-   ECC: c3467-   ECI: UTI89_C3274-   ECP: ECP_(—)2882-   ECV: APECO1_(—)3638-   ECW: EcE24377A_(—)3215(idi)-   ECX: EcHS_A3048-   STY: STY3195-   STT: t2957-   SPT: SPA2907(idi)-   SEC: SC2979(idi)-   STM: STM3039(idi)-   SFL: SF2875(idi)-   SFX: 53074-   SFV: SFV_(—)2937-   SSN: SSON_(—)3042 SSON_(—)3489(yhfK)-   SBO: SBO_(—)3103-   SDY: SDY_(—)3193-   ECA: ECA2789-   PLU: plu3987-   ENT: Ent638_(—)3307-   SPE: Spro_(—)2201-   VPA: VPA0278-   VFI: VF0403-   PPR: PBPRA0469(mvaD)-   PEN: PSEEN4850-   CBU: CBU_(—)0607(mvaD)-   CBD: COXBU7E912_(—)0619(mvaD)-   LPN: lpg2051-   LPF: lpl2029-   LPP: lpp2034-   TCX: Tcr_(—)1718-   HHA: Hhal_(—)1623-   DNO: DNO_(—)0798-   EBA: ebA5678 p2A143-   DVU: DVU1679(idi)-   DDE: Ddc_(—)1991-   LIP: LI1134-   BBA: Bd1626-   AFW: Anae109_(—)4082-   MXA: MXAN_(—)5021(fni)-   RPR: RP452-   RTY: RT0439(idi)-   RCO: RC0744-   RFE: RF_(—)0785(fni)-   RBE: RBE_(—)0731(fni)-   RAK: A1C_(—)04190-   RBO: A1I_(—)04755-   RCM: A1E_(—)02555-   RRI: A1G_(—)04195-   MLO: mlr6371-   RET: RHE_PD00245(ypd00046)-   XAU: Xaut_(—)4134-   SIL: SPO0131-   SIT: TM1040_(—)3442-   RSP: RSP_(—)0276-   RSH: Rsph17029_(—)1919-   RSQ: Rsph17025_(—)1019-   JAN: Jann_(—)0168-   RDE: RD1_(—)0147(idi)-   DSH: Dshi_(—)3527-   BSU: BG11440(ypgA)-   BAN: BA1520-   BAR: GBAA1520-   BAA: BA_(—)2041-   BAT: BAS1409-   BCE: BC1499-   BCA: BCE_(—)1626-   BCZ: BCZK1380(fni)-   BCY: Bcer98_(—)1222-   BTK: BT9727_(—)1381(fni)-   BTL: BALH_(—)1354-   BLI: BL02217(fni)-   BLD: BLi02426-   BAY: RBAM_(—)021020(fni)-   BPU: BPUM_(—)2020(fni)-   OIH: OB0537-   SAU: SA2136(fni)-   SAV: SAV2346(fni)-   SAM: MW2267(fni)-   SAR: SAR2431(fni)-   SAS: SAS2237-   SAC: SACOL2341(fni)-   SAB: SAB2225c(fni)-   SAA: SAUSA300_(—)2292(fni)-   SAO: SAOUHSC_(—)02623-   SEP: SE1925-   SER: SERP1937(fni-2)-   SHA: SH0712(fni)-   SSP: SSP0556-   LMO: 1mo1383-   LMF: LMOf2365_(—)1402(fni)-   LIN: lin1420-   LWE: lwel399(fni)-   LLA: L11083(yebB)-   LLC: LACR_(—)0457-   LLM: llmg_(—)0428(fni)-   SPY: SPy_(—)0879-   SPZ: M5005_Spy_(—)0685-   SPM: spyM18_(—)0940-   SPG: SpyM3_(—)0598-   SPS: SPs1255-   SPH: MGAS 10270_Spy0743-   SPI: MGAS10750_Spy0777-   SPJ: MGAS2096_Spy0756-   SPK: MGAS9429_Spy0740-   SPF: SpyM51123(fni)-   SPA: M6_Spy0702-   SPB: M28_Spy0665-   SPN: SP_(—)0384-   SPR: spr0341(fni)-   SPD: SPD_(—)0349(fni)-   SAG: SAG1323-   SAN: gbs1393-   SAK: SAK_(—)1354(fni)-   SMU: SMU.939-   STC: str0562(idi)-   STL: stu0562(idi)-   STE: STER_(—)0601-   SSA: SSA_(—)0336-   SGO: SGO_(—)0242-   LPL: lp_(—)1732(idi1)-   LJO: LJ1208-   LAC: LBA1171-   LSA: LSA0905(idi)-   LSL: LSL_(—)0682-   LDB: Ldb0996(fni)-   LBU: LBUL_(—)0903-   LBR: LVIS_(—)0861-   LCA: LSEI_(—)1493-   LGA: LGAS_(—)1036-   LRE: Lreu_(—)0912-   EFA: EF0901-   OOE: OEOE_(—)1103-   STH: STH1674-   CBE: Cbei_(—)3081-   DRM: Dred_(—)0474-   SWO: Swol_(—)1341-   MTA: Moth_(—)1328-   MTU: Rv1745c(idi)-   MTC: MT1787(idi)-   MBO: Mb1774c(idi)-   MBB: BCG_(—)1784c(idi)-   MPA: MAP3079c-   MAV: MAV_(—)3894(fni)-   MSM: MSMEG_(—)1057(fni) MSMEG_(—)2337(fni)-   MUL: MUL_(—)0380(idi2)-   MVA: Mvan_(—)1582 Mvan_(—)2176-   MGI: Mflv_(—)1842 Mflv_(—)4187-   MMC: Mmcs_(—)1954-   MKM: Mkms_(—)2000-   MJL: Mjls_(—)1934-   CGL: NCgl2223(cgl2305)-   CGB: cg2531(idi)-   CEF: CE2207-   CDI: DIP1730(idi)-   NFA: nfa19790 nfa22100-   RHA: RHA1_ro00239-   SCO: SCO6750(SC5F2A.33c)-   SMA: SAV1663(idi)-   LXX: Lxx23810(idi)-   CMI: CMM_(—)2889(idiA)-   AAU: AAur_(—)0321(idi)-   PAC: PPA2115-   FRA: Francci3_(—)4188-   FRE: Franean1_(—)5570-   FAL: FRAAL6504(idi)-   KRA: Krad_(—)3991-   SEN: SACE_(—)2627(idiB_(—)2) SACE_(—)5210(idi)-   STP: Strop_(—)4438-   SAQ: Sare_(—)4564 Sare_(—)4928-   RXY: Rxyl_(—)0400-   BBU: BB0684-   BGA: BG0707-   SYN: sll1556-   SYC: syc2161_c-   SYF: Synpcc7942_(—)1933-   CYA: CYA_(—)2395(fni)-   CYB: CYB_(—)2691(fni)-   TEL: tll1403-   ANA: all4591-   AVA: Ava_(—)2461 Ava_B0346-   TER: Tery_(—)1589-   SRU: SRU_(—)1900(idi)-   CHU: CHU_(—)0674(idi)-   GFO: GFO_(—)2363(idi)-   FJO: Fjoh_(—)0269-   FPS: FP1792(idi)-   CTE: CT0257-   CCH: Cag_(—)1445-   CPH: Cpha266_(—)0385-   PVI: Cvib_(—)1545-   PLT: Plut_(—)1764-   RRS: RoseRS_(—)2437-   RCA: Rcas_(—)2215-   HAU: Haur_(—)4687-   DRA: DR_(—)1087-   DGE: Dgeo_(—)1381-   TTH: TT_P0067-   TTJ: TTHB110-   MJA: MJ0862-   MMP: MMP0043-   MMQ: MmarC5_(—)1637-   MMX: MmarC6_(—)0906-   MMZ: MmarC7_(—)1040-   MAE: Maeo_(—)1184-   MVN: Mevan_(—)1058-   MAC: MA0604(idi)-   MBA: Mbar_A1419-   MMA: MM_(—)1764-   MBU: Mbur_(—)2397-   MTP: Mthe_(—)0474-   MHU: Mhun_(—)2888-   MLA: Mlab_(—)1665-   MEM: Memar_(—)1814-   MBN: Mboo_(—)2211-   MTH: MTH48-   MST: Msp_(—)0856(fni)-   MSI: Msm_(—)1441-   MKA: MK0776(lldD)-   AFU: AF2287-   HAL: VNG1818G(idi) VNG6081G(crt_(—)1) VNG6445G(crt_(—)2) VNG7060    VNG7149-   IImMA: rrnAC3484(idi)-   HWA: HQ2772A(idiA) HQ2847A(idiB)-   NPH: NP0360A(idiB_(—)1) NP4826A(idiA) NP5124A(idiB_(—)2)-   TAC: Ta0102-   TVO: TVN0179-   PTO: PTO0496-   PHO: PH1202-   PAB: PAB1662-   PFU: PF0856-   TKO: TK1470-   RCI: LRC397(fni)-   APE: APE_(—)1765.1-   SMR: Smar_(—)0822-   IHO: Igni_(—)0804-   HBU: Hbut_(—)0539-   SSO: SSO0063-   STO: ST2059-   SAI: Saci_(—)0091-   MSE: Msed_(—)2136-   PAI: PAE0801-   PIS: Pisl_(—)1093-   PCL: Pcal_(—)0017-   PAS: Pars_(—)0051-   TPE: Tpen_(—)0272    Exemplary Isoprene Synthase Nucleic Acids and Polypeptides-   Genbank Accession Nos.-   AY341431-   AY316691-   AY279379-   AJ457070-   AY182241

1. Recombinant cells capable of producing isoprene, said cellscomprising: (a) one or more non-modified heterologous nucleic acidsencoding feedback-resistant mevalonate kinase polypeptides, (b) one ormore heterologous nucleic acids encoding an isoprene synthasepolypeptide or one or more additional copies of an endogenous nucleicacid encoding an isoprene synthase polypeptide, and (c) one or morenucleic acids encoding a mevalonate (MVA) pathway polypeptide, whereinsaid cells produce isoprene.
 2. The cells of claim 1, wherein thefeedback-resistant mevalonate kinase is archaeal mevalonate kinase. 3.The cells of claim 1, wherein the (c) one or more nucleic acids encodinga mevalonate (MVA) pathway polypeptide is heterologous.
 4. The cells ofclaim 1, wherein the (c) one or more nucleic acids encoding a mevalonate(MVA) pathway polypeptide is endogenous.
 5. The cells of claim 2,wherein the feedback-resistant archaeal mevalonate kinase polypeptide isM. mazei mevalonate kinase.
 6. The cells of claim 1, wherein the cellsproduce greater than about 400 nmole/g_(wcm)/hr of isoprene.
 7. Thecells of claim 1, wherein the isoprene synthase polypeptide is fromPueraria or Populus or a hybrid, Populus alba×Populus tremula.
 8. Thecells of claim 7, wherein the isoprene synthase polypeptide is selectedfrom the group consisting of Pueraria montana or Pueraria lobata,Populus tremuloides, Populus alba, Populus nigra, and Populustrichocarpa.
 9. The cells of claim 1, wherein the cells are Streptomycescells, Escherichia cells, Pantoea cells, Trichoderma cells, orAspergillus cells.
 10. The cells of claim 9, wherein the cells areselected from the group consisting of Bacillus subtilis, Streptomyceslividans, Streptomyces coelicolor, Streptomyces griseus, Escherichiacoli, Pantoea citrea, Trichoderma reesei, Aspergillus oryzae andAspergillus niger, Saccharomyces cerevisieae and Yarrowia lipolytica.11. A composition for producing isoprene comprising cells of claim 1.12. The cells of claim 1 wherein the one or more nucleic acids encodinga mevalonate (MVA) pathway polypeptide is selected from the groupconsisting of acetyl-CoA acetyltransferase (AA-CoA thiolase)polypeptide, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)polypeptide, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAreductase) polypeptide, mevalonate kinase (MVK) polypeptide,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonatedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides.
 13. Thecells of claim 1 wherein the one or more nucleic acids encoding amevalonate (MVA) pathway polypeptide encodes for upper MVA pathwaypolypeptide selected from the group consisting of AA-CoA thiolase,HMG-CoA synthase, and HMG-CoA reductase polypeptide.
 14. The cells ofclaim 1 wherein the one or more nucleic acids encoding a mevalonate(MVA) pathway polypeptide encodes for lower MVA pathway polypeptideselected from the group consisting of MVK, PMK, MVD, and IDI(Isopentenyl-diphosphate delta isomerase) polypeptide.
 15. The cells ofclaim 1 wherein the feedback-resistant mevalonate kinase polypeptide hasa K_(i)≧500 μM DMAPP.
 16. The cells of claim 1 wherein thefeedback-resistant mevalonate kinase polypeptide has a K_(i)≧20 μM GPP.17. The cells of claim 1 wherein the feedback-resistant mevalonatekinase polypeptide has a K_(i)≧20 μM FPP.
 18. The cells of claim 1further comprising one or more nucleic acids encoding a non-mevalonate(DXP) pathway polypeptide.
 19. The cells of claim 1, wherein the cellsare gram-positive bacterial cells, gram-negative bacterial cells, fungalcells, filamentous fungal cells, or yeast cells.